Reagents and methods useful for detecting diseases of the prostate

ABSTRACT

A set of contiguous and partially overlapping RNA sequences and polypeptides encoded thereby, designated as PS190 and transcribed from prostate tissue is described. These sequences are useful for the detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining the predisposition of an individual to diseases and conditions of the prostate, such as prostate cancer. Also provided are antibodies which specifically bind to PS190-encoded polypeptide or protein, and agonists or inhibitors which prevent action of the tissue-specific PS190 polypeptide, which molecules are useful for the therapeutic treatment of prostate diseases, tumors or metastases.

This application is a continuation-in-part of Ser. No. 08/926,509 filedon Sep. 9, 1997 now abandoned.

BACKGROUND OF THE INVENTION

The invention relates generally to detecting diseases of the prostate,and more particularly, relates to reagents such as polynucleotidesequences and the polypeptide sequences encoded thereby, as well asmethods which utilize these sequences, which are useful for detecting,diagnosing, staging, monitoring, prognosticating, preventing ortreating, or determining predisposition to diseases or conditions of theprostate such as prostate cancer.

Prostate cancer is the most common form of cancer occurring in males inthe United States, with projections of 334,500 new cases diagnosed and41,800 related deaths predicted to occur during 1997 (American CancerSociety). Prostate cancer also has shown the largest increase inincidence as compared to other types of cancer, increasing 142% from1992 to 1996.

Procedures used for detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining predispositionto diseases or conditions of the prostate such as prostate cancer are ofcritical importance to the outcome of the patient. For example, patientsdiagnosed with localized prostate cancer have greater than a 90%five-year survival rate compared to a rate of 25 to 31% for patientsdiagnosed with distant metastasis. (American Cancer Society statistics).A diagnostic procedure for early detection of prostate cancer should,therefore, specifically detect this disease and be capable of detectingthe presence of prostate cancer before symptoms appear.

Such procedures could include assays based upon the appearance ofvarious disease markers in test samples such as blood, plasma, serum, orurine obtained by minimally invasive procedures which are detectable byimmunological methods. These procedures would provide information to aidthe physician in managing the patient with disease of the prostate andat low cost to the patient. Markers such as the prostate specificantigen (PSA) exist and are used clinically for screening patients forprostate cancer. Elevated levels of PSA protein in serum can be used asa marker in the early detection of prostate cancer in asymptomatic men.G. E. Hanks, et al., In: Cancer: Principles and Practice of OncologyVol. 1, Fourth Edition, pp. 1073-1113, Philadelphia, Pa.: J.B.Lippincott Co. (1993.). PSA normally is secreted by the prostate at highlevels into the seminal fluid, but is present in very low levels in theblood of men with normal prostates. However, in patients with diseasesof the prostate including benign prostatic hyperplasia (BPH) andadenocarcinoma of the prostate, the level of PSA can be markedlyelevated in the blood and thus be useful as an indicator of prostatedisease. PSA, however, cannot differentiate between BPH and prostatecancer, which reduces its specificity as a marker for prostate cancer.M. K. Schwartz, et al., In: Cancer: Principles and Practice of Oncology,Vol. 1, Fourth Edition, pp. 531-542, Philadelphia, Pa.: J.B. LippincottCo. 1993. New markers which are more specific for prostate cancer thuswould be beneficial in the initial detection of this disease.

A critical step in managing patients with prostate cancer is thepresurgical staging of the cancer to provide prognostic value andcriteria for designing optimal therapy. Improved procedures foraccurately staging prostate cancer prior to surgery are needed. Onestudy demonstrated that current methods of staging prostate cancer priorto surgery were incorrect approximately fifty percent (50%) of the time.F. Labrie, et al., Urology 44 (Symposium Issue): 29-37 (1994). Prostatecancer management also could be improved by utilizing new markers foundin an inappropriate body compartment. Such markers could be mRNA orprotein markers expressed by cells originating from the primary prostatetumor but residing in blood, bone marrow or lymph nodes and could besensitive indicators for metastasis to these distal organs. For example,in patients with metastatic prostate cancer, PSA protein has beendetected by immunohistochemical techniques in bone marrow, and PSA mRNAhas been detected by RT-PCR in cells of blood, lymph nodes and bonemarrow. K. Pantel, et al., Onkologie 18: 394-401 (1995).

New markers which could predict the biologic behavior of early prostatecancers would also be of significant value. Early prostate cancers thatthreaten or will threaten the life of the patient are more clinicallyimportant than those that do not or will not be a threat. G. E. Hanks,supra. A need therefore exists for new markers which can differentiatebetween the clinically important and unimportant prostate cancers. Suchmarkers would allow the clinician to accurately identify and effectivelytreat early cancers localized to the prostate which could otherwisemetastasize and kill the patient. Further, if one could show that such amarker characteristic of aggressive cancer was absent, the patient couldbe spared expensive and non-beneficial treatment.

It also would be beneficial to find a prostate associated marker whichis more sensitive in detecting recurrence of prostate cancer than PSAand which is not affected by androgens. To date, PSA has proven to bethe most sensitive marker for detecting recurrent disease. However, insome cases tumor progression occurs without PSA elevation due tohormonal therapy utilized for treating the cancer. Although the decreasein androgen results in a concomitant decrease in PSA, it does notnecessarily reflect a decrease in tumor metastasis. This complication isthe result of androgen-stimulated PSA expression. Part of the decline inPSA observed after androgen ablation is due not to tumor cell death butto diminished PSA expression. G. E. Hanks, supra.

It therefore would be advantageous to provide specific methods andreagents for detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining predispositionto diseases and conditions of the prostate, or to indicate possiblepredisposition to these conditions. Such methods would include assayinga test sample for products of a gene which are overexpressed in prostatediseases and conditions such as cancer. Such methods may also includeassaying a test sample for products of a gene alteration associated withprostate disease or condition. Such methods may further include assayinga test sample for products of a gene whose distribution among thevarious tissues and compartments of the body have been altered by aprostate-associated disease or condition such as cancer. Useful reagentsinclude polynucleotides, or fragments thereof, which may be used indiagnostic methods such as reverse transcriptase-polymerase chainreaction (RT-PCR), PCR, or hybridization assays of mRNA extracted frombiopsied tissue, blood or other test samples. Other useful reagentsinclude polypeptides or proteins which are the translation products ofsuch mRNAs, and antibodies directed against these polypeptides orproteins. Drug treatment or gene therapy for diseases or conditions ofthe prostate can then be based on these identified gene sequences ortheir expressed proteins and efficacy of any particular therapy can bemonitored. Furthermore, it would be advantageous to have availablealternative, non-surgical diagnostic methods capable of detecting earlystage prostate disease such as cancer.

SUMMARY OF THE INVENTION

The present invention provides a method of detecting a target PS190polynucleotide in a test sample which comprises contacting the testsample with at least one PS190-specific polynucleotide and detecting thepresence of the target PS190 polynucleotide in the test sample. ThePS190-specific polynucleotide has at least 50% identity with apolynucleotide selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof. Also, the PS190-specific polynucleotide may beattached to a solid phase prior to performing the method.

The present invention also provides a method for detecting PS190 mRNA ina test sample, which comprises performing reverse transcription (RT)with at least one primer in order to produce cDNA, amplifying the cDNAso obtained using PS190 oligonucleotides as sense and antisense primersto obtain PS190 amplicon, and detecting the presence of the PS190amplicon as an indication of the presence of PS190 mRNA in the testsample, wherein the PS190 oligonucleotides have at least 50% identity toa sequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof. Amplification can be performed by the polymerasechain reaction. Also, the test sample can be reacted with a solid phaseprior to performing the method, prior to amplification or prior todetection. This reaction can be a direct or an indirect reaction.Further, the detection step can comprise utilizing a detectable labelcapable of generating a measurable signal. The detectable label can beattached to a solid phase.

The present invention further provides a method of detecting a targetPS190 polynucleotide in a test sample suspected of containing targetPS190 polynucleotides, which comprises (a) contacting the test samplewith at least one PS190 oligonucleotide as a sense primer and at leastone PS190 oligonucleotide as an anti-sense primer, and amplifying sameto obtain a first stage reaction product; (b) contacting the first stagereaction product with at least one “other” PS190 oligonucleotide toobtain a second stage reaction product, with the proviso that the“other” PS190 oligonucleotide is located 3′ to the PS190oligonucleotides utilized in step (a) and is complementary to the firststage reaction product; and (c) detecting the second stage reactionproduct as an indication of the presence of a target PS190polynucleotide in the test sample. The PS190 oligonucleotides selectedas reagents in the method have at least 50% identity to a sequenceselected from the group consisting of SEQUENCE ID NO 1, SEQUENCE ID NO2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments or complementsthereof. Amplification may be performed by the polymerase chainreaction. The test sample can be reacted either directly or indirectlywith a solid phase prior to performing the method, or prior toamplification, or prior to detection. The detection step also comprisesutilizing a detectable label capable of generating a measurable signal;further, the detectable label can be attached to a solid phase. Testkits useful for detecting target PS190 polynucleotides in a test sampleare also provided which comprise a container containing at least onePS190-specific polynucleotide selected from the group consisting ofSEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4,and fragments or complements thereof. These test kits further comprisecontainers with tools useful for collecting test samples (such as, forexample, blood, urine, saliva and stool). Such tools include lancets andabsorbent paper or cloth for collecting and stabilizing blood; swabs forcollecting and stabilizing saliva; and cups for collecting andstabilizing urine or stool samples. Collection materials, such aspapers, cloths, swabs, cups and the like, may optionally be treated toavoid denaturation or irreversible adsorption of the sample. Thecollection materials also may be treated with or contain preservatives,stabilizers or antimicrobial agents to help maintain the integrity ofthe specimens.

The present invention provides a purified polynucleotide or fragmentthereof derived from a PS190 gene. The purified polynucleotide iscapable of selectively hybridizing to the nucleic acid of the PS190gene, or a complement thereof. The polynucleotide has at least 50%identity to a polynucleotide selected from the group consisting ofSEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4,and fragments or complements thereof. Further, the purifiedpolynucleotide can be produced by recombinant and/or synthetictechniques. The purified recombinant polynucleotide can be containedwithin a recombinant vector. The invention further comprises a host celltransfected with said vector.

The present invention further provides a recombinant expression systemcomprising a nucleic acid sequence that includes an open reading framederived from PS190. The nucleic acid sequence has at least 50% identitywith a sequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof. The nucleic acid sequence is operably linked to acontrol sequence compatible with a desired host. Also provided is a celltransfected with this recombinant expression system.

The present invention also provides a polypeptide encoded by PS190. Thepolypeptide can be produced by recombinant technology, provided inpurified form, or produced by synthetic techniques. The polypeptidecomprises an amino acid sequence which has at least 50% identity to anamino acid sequence selected from the group consisting of SEQUENCE ID NO9, SEQUENCE ID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO 12, SEQUENCE IDNO 13, SEQUENCE ID NO 14, and fragments thereof.

Also provided is an antibody which specifically binds to at least onePS190 epitope. The antibody can be a polyclonal or monoclonal antibody.The epitope is derived from an amino acid sequence selected from thegroup consisting of SEQUENCE ID NO 9, SEQUENCE ID NO 1, SEQUENCE ID NO11, SEQUENCE ID NO 12, SEQUENCE ID NO 13, SEQUENCE ID NO 14, andfragments thereof. Assay kits for determining the presence of PS190antigen or anti-PS190 antibody in a test sample are also included. Inone embodiment, the assay kits comprise a container containing at leastone PS190 polypeptide having at least 50% identity to an amino acidsequence selected from the group consisting of SEQUENCE ID NO 9,SEQUENCE ID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO 12, SEQUENCE ID NO13, SEQUENCE ID NO 14, and fragments thereof. Further, the test kit cancomprise a container with tools useful for collecting test samples (suchas blood, urine, saliva and stool). Such tools include lancets andabsorbent paper or cloth for collecting and stabilizing blood; swabs forcollecting and stabilizing saliva; and cups for collecting andstabilizing urine or stool samples. Collection materials such as,papers, cloths, swabs, cups and the like, may optionally be treated toavoid denaturation or irreversible adsorption of the sample. Thesecollection materials also may be treated with or contain preservatives,stabilizers or antimicrobial agents to help maintain the integrity ofthe specimens. Also, the polypeptide can be attached to a solid phase.

Another assay kit for determining the presence of PS190 antigen oranti-PS190 antibody in a test sample comprises a container containing anantibody which specifically binds to a PS190 antigen, wherein the PS190antigen comprises at least one PS190-encoded epitope. The PS190 antigenhas at least about 60% sequence similarity to a sequence of aPS190-encoded antigen selected from the group consisting of SEQUENCE IDNO 9, SEQUENCE ID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO 12, SEQUENCEID NO 13, SEQUENCE ID NO 14, and fragments thereof. These test kits canfurther comprise containers with tools useful for collecting testsamples (such as blood, urine, saliva and stool). Such tools includelancets and absorbent paper or cloth for collecting and stabilizingblood; swabs for collecting and stabilizing saliva; cups for collectingand stabilizing urine or stool samples. Collection materials, papers,cloths, swabs, cups and the like, may optionally be treated to avoiddenaturation or irreversible adsorption of the sample. These collectionmaterials also may be treated with, or contain, preservatives,stabilizers or antimicrobial agents to help maintain the integrity ofthe specimens. The antibody can be attached to a solid phase.

A method for producing a polypeptide which contains at least one epitopeof PS190 is provided, which method comprises incubating host cellstransfected with an expression vector. This vector comprises apolynucleotide sequence encoding a polypeptide, wherein the polypeptidecomprises an amino acid sequence having at least 50% identity to a PS190amino acid sequence selected from the group consisting of SEQUENCE ID NO9, SEQUENCE ID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO 12, SEQUENCE IDNO 13, SEQUENCE ID NO 14, and fragments thereof.

A method for detecting PS190 antigen in a test sample suspected ofcontaining PS190 antigen also is provided. The method comprisescontacting the test sample with an antibody or fragment thereof whichspecifically binds to at least one epitope of a PS190 antigen, for atime and under conditions sufficient for the formation ofantibody/antigen complexes; and detecting the presence of such complexescontaining the antibody as an indication of the presence of PS190antigen in the test sample. The antibody can be attached to a solidphase and be either a monoclonal or polyclonal antibody. Furthermore,the antibody specifically binds to at least one PS190 antigen selectedfrom the group consisting of SEQUENCE ID NO 9, SEQUENCE ID NO 10,SEQUENCE ID NO 11, SEQUENCE ID NO 12, SEQUENCE ID NO 13, SEQUENCE ID NO14, and fragments thereof.

Another method is provided which detects antibodies which specificallybind to PS190 antigen in a test sample suspected of containing theseantibodies. The method comprises contacting the test sample with apolypeptide which contains at least one PS190 epitope, wherein the PS190epitope comprises an amino acid sequence having at least 50% identitywith an amino acid sequence encoded by a PS190 polynucleotide, or afragment thereof. Contacting is carried out for a time and underconditions sufficient to allow antigen/antibody complexes to form. Themethod further entails detecting complexes which contain thepolypeptide. The polypeptide can be attached to a solid phase. Further,the polypeptide can be a recombinant protein or a synthetic peptidehaving at least 50% identity to an amino acid sequence selected from thegroup consisting of SEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE ID NO11, SEQUENCE ID NO 12, SEQUENCE ID NO 13, SEQUENCE ID NO 14, andfragments thereof.

The present invention provides a cell transfected with a PS190 nucleicacid sequence that encodes at least one epitope of a PS190 antigen, orfragment thereof. The nucleic acid sequence is selected from the groupconsisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3,SEQUENCE ID NO 4, and fragments or complements thereof.

A method for producing antibodies to PS190 antigen also is provided,which method comprises administering to an individual an isolatedimmunogenic polypeptide or fragment thereof, wherein the isolatedimmunogenic polypeptide comprises at least one PS190 epitope in anamount sufficient to produce an immune response. The isolated,immunogenic polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE IDNO 11, SEQUENCE ID NO 12, SEQUENCE ID NO 13, SEQUENCE ID NO 14, andfragments thereof.

Another method for producing antibodies which specifically bind to PS190antigen is disclosed, which method comprises administering to a mammal aplasmid comprising a nucleic acid sequence which encodes at least onePS190 epitope derived from an amino acid sequence selected from thegroup consisting of SEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE ID NO11, SEQUENCE ID NO 12, SEQUENCE ID NO 13, SEQUENCE ID NO 14, andfragments thereof.

Also provided is a composition of matter that comprises a PS190polynucleotide of at least about 10-12 nucleotides having at least 50%identity to a polynucleotide selected from the group consisting ofSEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4,and fragments or complements thereof. The PS190 polynucleotide encodesan amino acid sequence having at least one PS190 epitope. Anothercomposition of matter provided by the present invention comprises apolypeptide with at least one PS190 epitope of about 8-10 amino acids.The polypeptide comprises an amino acid sequence having at least 50%identity to an amino acid sequence selected from the group consisting ofSEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO12, SEQUENCE ID NO 13, SEQUENCE ID NO 14, and fragments thereof. Alsoprovided is a gene, or fragment thereof, coding for a PS190 polypeptidewhich has at least 50% identity to SEQUENCE ID NO 9, and a gene, or afragment thereof, comprising DNA having at least 50% identity toSEQUENCE ID NO 4.

BRIEF DESCRIPTION OF THE DRAWINGS

There are no drawings in this case.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a gene or a fragment thereof which codesfor a PS190 polypeptide having at least about 50% identity to SEQUENCEID NO 9. The present invention further encompasses a PS190 gene or afragment thereof comprising DNA which has at least about 50% identity toSEQUENCE ID NO 4.

The present invention provides methods for assaying a test sample forproducts of a prostate tissue gene designated as PS190, which comprisesmaking cDNA from mRNA in the test sample, and detecting the cDNA as anindication of the presence of prostate tissue gene PS190. The method mayinclude an amplification step, wherein one or more portions of the mRNAfrom PS190 corresponding to the gene or fragments thereof, is amplified.Methods also are provided for assaying for the translation products ofPS190. Test samples which may be assayed by the methods provided hereininclude tissues, cells, body fluids and secretions. The presentinvention also provides reagents such as oligonucleotide primers andpolypeptides which are useful in performing these methods.

Portions of the nucleic acid sequences disclosed herein are useful asprimers for the reverse transcription of RNA or for the amplification ofcDNA; or as probes to determine the presence of certain mRNA sequencesin test samples. Also disclosed are nucleic acid sequences which permitthe production of encoded polypeptide sequences which are useful asstandards or reagents in diagnostic immunoassays, as targets forpharmaceutical screening assays and/or as components or as target sitesfor various therapies. Monoclonal and polyclonal antibodies directedagainst at least one epitope contained within these polypeptidesequences are useful as delivery agents for therapeutic agents as wellas for diagnostic tests and for screening for diseases or conditionsassociated with PS190, especially prostate cancer. Isolation ofsequences of other portions of the gene of interest can be accomplishedutilizing probes or PCR primers derived from these nucleic acidsequences. This allows additional probes of the mRNA or cDNA of interestto be established, as well as corresponding encoded polypeptidesequences. These additional molecules are useful in detecting,diagnosing, staging, monitoring, prognosticating, preventing ortreating, or determining the predisposition to, diseases and conditionsof the prostate such as prostate cancer, characterized by PS190, asdisclosed herein.

Techniques for determining amino acid sequence “similarity” arewell-known in the art. In general, “similarity” means the exact aminoacid to amino acid comparison of two or more polypeptides at theappropriate place, where amino acids are identical or possess similarchemical and/or physical properties such as charge or hydrophobicity. Aso-termed “percent similarity” then can be determined between thecompared polypeptide sequences. Techniques for determining nucleic acidand amino acid sequence identity also are well known in the art andinclude determining the nucleotide sequence of the mRNA for that gene(usually via a cDNA intermediate) and determining the amino acidsequence encoded thereby, and comparing this to a second amino acidsequence. In general, “identity” refers to an exact nucleotide tonucleotide or amino acid to amino acid correspondence of twopolynucleotides or polypeptide sequences, respectively. Two or morepolynucleotide sequences can be compared by determining their “percentidentity.” Two or more amino acid sequences likewise can be compared bydetermining their “percent identity.” The programs available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.), for example, the GAP program, arecapable of calculating both the identity between two polynucleotides andthe identity and similarity between two polypeptide sequences,respectively. Other programs for calculating identity or similaritybetween sequences are known in the art.

The compositions and methods described herein will enable theidentification of certain markers as indicative of a prostate tissuedisease or condition; the information obtained therefrom will aid in thedetecting, diagnosing, staging, monitoring, prognosticating, preventingor treating, or determining diseases or conditions associated withPS190, especially prostate cancer. Test methods include, for example,probe assays which utilize the sequence(s) provided herein and whichalso may utilize nucleic acid amplification methods such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), andhybridization. In addition, the nucleotide sequences provided hereincontain open reading frames from which an immunogenic epitope may befound. This epitope is believed to be unique to the disease state orcondition associated with PS190. It also is thought that thepolynucleotides or polypeptides and protein encoded by the PS190 geneare useful as a marker. This marker is either elevated in disease suchas prostate cancer, altered in disease such as prostate cancer, orpresent as a normal protein but appearing in an inappropriate bodycompartment. The uniqueness of the epitope may be determined by (i) itsimmunological reactivity and specificity with antibodies directedagainst proteins and polypeptides encoded by the PS190 gene, and (ii)its nonreactivity with any other tissue markers. Methods for determiningimmunological reactivity are well-known and include but are not limitedto, for example, radioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), hemagglutination (HA), fluorescence polarizationimmunoassay (FPIA), chemiluminescent immunoassay (CLIA) and others.Several examples of suitable methods are described herein.

Unless otherwise stated, the following terms shall have the followingmeanings:

A polynucleotide “derived from” or “specific for” a designated sequencerefers to a polynucleotide sequence which comprises a contiguoussequence of approximately at least about 6 nucleotides, preferably atleast about 8 nucleotides, more preferably at least about 10-12nucleotides, and even more preferably at least about 15-20 nucleotidescorresponding, i.e., identical or complementary to, a region of thedesignated nucleotide sequence. The sequence may be complementary oridentical to a sequence which is unique to a particular polynucleotidesequence as determined by techniques known in the art. Comparisons tosequences in databanks, for example, can be used as a method todetermine the uniqueness of a designated sequence. Regions from whichsequences may be derived, include but are not limited to, regionsencoding specific epitopes, as well as non-translated and/ornon-transcribed regions.

The derived polynucleotide will not necessarily be derived physicallyfrom the nucleotide sequence of interest under study, but may begenerated in any manner, including but not limited to chemicalsynthesis, replication, reverse transcription or transcription, which isbased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. As such, it mayrepresent either a sense or an antisense orientation of the originalpolynucleotide. In addition, combinations of regions corresponding tothat of the designated sequence may be modified in ways known in the artto be consistent with the intended use.

A “fragment” of a specified polynucleotide refers to a polynucleotidesequence which comprises a contiguous sequence of approximately at leastabout 6 nucleotides, preferably at least about 8 nucleotides, morepreferably at least about 10-12 nucleotides, and even more preferably atleast about 15-20 nucleotides corresponding, i.e., identical orcomplementary to, a region of the specified nucleotide sequence.

The term “primer” denotes a specific oligonucleotide sequence which iscomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. A primer serves as an initiation pointfor nucleotide polymerization catalyzed by either DNA polymerase, RNApolymerase or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotideanalog segment, e.g., PNA as defined hereinbelow) which can be used toidentify a specific polynucleotide present in samples bearing thecomplementary sequence.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences which areimmunologically identifiable with a polypeptide encoded by the sequence.Thus, a “polypeptide,” “protein,” or “amino acid” sequence has at leastabout 50% identity, preferably about 60% identity, more preferably about75-85% identity, and most preferably about 90-95% or more identity to aPS190 amino acid sequence. Further, the PS190 “polypeptide,” “protein,”or “amino acid” sequence may have at least about 60% similarity,preferably at least about 75% similarity, more preferably about 85%similarity, and most preferably about 95% or more similarity to apolypeptide or amino acid sequence of PS190. This amino acid sequencecan be selected from the group consisting of SEQUENCE ID NO 9, SEQUENCEID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO 12, SEQUENCE ID NO 13,SEQUENCE ID NO 14, and fragments thereof.

A “recombinant polypeptide,” “recombinant protein,” or “a polypeptideproduced by recombinant techniques,” which terms may be usedinterchangeably herein, describes a polypeptide which by virtue of itsorigin or manipulation is not associated with all or a portion of thepolypeptide with which it is associated in nature and/or is linked to apolypeptide other than that to which it is linked in nature. Arecombinant or encoded polypeptide or protein is not necessarilytranslated from a designated nucleic acid sequence. It also may begenerated in any manner, including chemical synthesis or expression of arecombinant expression system.

The term “synthetic peptide” as used herein means a polymeric form ofamino acids of any length, which may be chemically synthesized bymethods well-known to the routineer. These synthetic peptides are usefulin various applications.

The term “polynucleotide” as used herein means a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes double- and single-stranded DNA,as well as double- and single-stranded RNA. It also includesmodifications, such as methylation or capping and unmodified forms ofthe polynucleotide. The terms “polynucleotide,” “oligomer,”“oligonucleotide,” and “oligo” are used interchangeably herein.

“A sequence corresponding to a cDNA” means that the sequence contains apolynucleotide sequence that is identical or complementary to a sequencein the designated DNA. The degree (or “percent”) of identity orcomplementarity to the cDNA will be approximately 50% or greater,preferably at least about 70% or greater, and more preferably at leastabout 90% or greater. The sequence that corresponds to the identifiedcDNA will be at least about 50 nucleotides in length, preferably atleast about 60 nucleotides in length, and more preferably at least about70 nucleotides in length. The correspondence between the gene or genefragment of interest and the cDNA can be determined by methods known inthe art and include, for example, a direct comparison of the sequencedmaterial with the cDNAs described, or hybridization and digestion withsingle strand nucleases, followed by size determination of the digestedfragments.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

“Purified polypeptide” or “purified protein” means a polypeptide ofinterest or fragment thereof which is essentially free of, e.g.,contains less than about 50%, preferably less than about 70%, and morepreferably less than about 90%, cellular components with which thepolypeptide of interest is naturally associated. Methods for purifyingpolypeptides of interest are known in the art.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, which is separated from some orall of the coexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

“Polypeptide” and “protein” are used interchangeably herein and indicateat least one molecular chain of amino acids linked through covalentand/or non-covalent bonds. The terms do not refer to a specific lengthof the product. Thus peptides, oligopeptides and proteins are includedwithin the definition of polypeptide. The terms includepost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Inaddition, protein fragments, analogs, mutated or variant proteins,fusion proteins and the like are included within the meaning ofpolypeptide.

A “fragment” of a specified polypeptide refers to an amino acid sequencewhich comprises at least about 3-5 amino acids, more preferably at leastabout 8-10 amino acids, and even more preferably at least about 15-20amino acids derived from the specified polypeptide.

“Recombinant host cells,” “host cells,” “cells,” “cell lines,” “cellcultures,” and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vector orother transferred DNA, and include the original progeny of the originalcell which has been transfected.

As used herein “replicon” means any genetic element, such as a plasmid,a chromosome or a virus, that behaves as an autonomous unit ofpolynucleotide replication within a cell.

A “vector” is a replicon in which another polynucleotide segment isattached, such as to bring about the replication and/or expression ofthe attached segment.

The term “control sequence” refers to a polynucleotide sequence which isnecessary to effect the expression of a coding sequence to which it isligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, such control sequences generally includea promoter, a ribosomal binding site and terminators; in eukaryotes,such control sequences generally include promoters, terminators and, insome instances, enhancers. The term “control sequence” thus is intendedto include at a minimum all components whose presence is necessary forexpression, and also may include additional components whose presence isadvantageous, for example, leader sequences.

“Operably linked” refers to a situation wherein the components describedare in a relationship permitting them to function in their intendedmanner. Thus, for example, a control sequence “operably linked” to acoding sequence is ligated in such a manner that expression of thecoding sequence is achieved under conditions compatible with the controlsequence.

The term “open reading frame” or “ORF” refers to a region of apolynucleotide sequence which encodes a polypeptide. This region mayrepresent a portion of a coding sequence or a total coding sequence.

A “coding sequence” is a polynucleotide sequence which is transcribedinto mRNA and translated into a polypeptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include, but is not limited to, mRNA, cDNA and recombinantpolynucleotide sequences.

The term “immunologically identifiable with/as” refers to the presenceof epitope(s) and polypeptide(s) which also are present in and areunique to the designated polypeptide(s). Immunological identity may bedetermined by antibody binding and/or competition in binding. Thesetechniques are known to the routineer and also are described herein. Theuniqueness of an epitope also can be determined by computer searches ofknown data banks, such as GenBank, for the polynucleotide sequence whichencodes the epitope and by amino acid sequence comparisons with otherknown proteins.

As used herein, “epitope” means an antigenic determinant of apolypeptide or protein. Conceivably, an epitope can comprise three aminoacids in a spatial conformation which is unique to the epitope.Generally, an epitope consists of at least five such amino acids andmore usually, it consists of at least eight to ten amino acids. Methodsof examining spatial conformation are known in the art and include, forexample, x-ray crystallography and two-dimensional nuclear magneticresonance.

A “conformational epitope” is an epitope that is comprised of specificjuxtaposition of amino acids in an immunologically recognizablestructure, such amino acids being present on the same polypeptide in acontiguous or non-contiguous order or present on different polypeptides.

A polypeptide is “immunologically reactive” with an antibody when itbinds to an antibody due to antibody recognition of a specific epitopecontained within the polypeptide. Immunological reactivity may bedetermined by antibody binding, more particularly, by the kinetics ofantibody binding, and/or by competition in binding using ascompetitor(s) a known polypeptide(s) containing an epitope against whichthe antibody is directed. The methods for determining whether apolypeptide is immunologically reactive with an antibody are known inthe art.

As used herein, the term “immunogenic polypeptide containing an epitopeof interest” means naturally occurring polypeptides of interest orfragments thereof, as well as polypeptides prepared by other means, forexample, by chemical synthesis or the expression of the polypeptide in arecombinant organism.

The term “transfection” refers to the introduction of an exogenouspolynucleotide into a prokaryotic or eucaryotic host cell, irrespectiveof the method used for the introduction. The term “transfection” refersto both stable and transient introduction of the polynucleotide, andencompasses direct uptake of polynucleotides, transformation,transduction, and f-mating. Once introduced into the host cell, theexogenous polynucleotide may be maintained as a non-integrated replicon,for example, a plasmid, or alternatively, may be integrated into thehost genome.

“Treatment” refers to prophylaxis and/or therapy.

The term “individual” as used herein refers to vertebrates, particularlymembers of the mammalian species and includes, but is not limited to,domestic animals, sports animals, primates and humans; more particularlythe term refers to humans.

The term “sense strand” or “plus strand” (or “+”) as used herein denotesa nucleic acid that contains the sequence that encodes the polypeptide.The term “antisense strand” or “minus strand” (or “−”) denotes a nucleicacid that contains a sequence that is complementary to that of the“plus” strand.

The term “test sample” refers to a component of an individual's bodywhich is the source of the analyte (such as, antibodies of interest orantigens of interest). These components are well known in the art. Atest sample is typically anything suspected of containing a targetsequence. Test samples can be prepared using methodologies well known inthe art such as by obtaining a specimen from an individual and, ifnecessary, disrupting any cells contained thereby to release targetnucleic acids. These test samples include biological samples which canbe tested by the methods of the present invention described herein andinclude human and animal body fluids such as whole blood, serum, plasma,cerebrospinal fluid, sputum, bronchial washing, bronchial aspirates,urine, lymph fluids and various external secretions of the respiratory,intestinal and genitourinary tracts, tears, saliva, milk, white bloodcells, myelomas and the like; biological fluids such as cell culturesupernatants; tissue specimens which may be fixed; and cell specimenswhich may be fixed.

“Purified product” refers to a preparation of the product which has beenisolated from the cellular constituents with which the product isnormally associated and from other types of cells which may be presentin the sample of interest.

“PNA” denotes a “peptide nucleic acid analog” which may be utilized in aprocedure such as an assay described herein to determine the presence ofa target. “MA” denotes a “morpholino analog” which may be utilized in aprocedure such as an assay described herein to determine the presence ofa target. See, for example, U.S. Pat. No. 5,378,841, which isincorporated herein by reference. PNAs are neutrally charged moietieswhich can be directed against RNA targets or DNA. PNA probes used inassays in place of, for example, the DNA probes of the presentinvention, offer advantages not achievable when DNA probes are used.These advantages include manufacturability, large scale labeling,reproducibility, stability, insensitivity to changes in ionic strengthand resistance to enzymatic degradation which is present in methodsutilizing DNA or RNA. These PNAs can be labeled with (“attached to”)such signal generating compounds as fluorescein, radionucleotides,chemiluminescent compounds and the like. PNAs or other nucleic acidanalogs such as MAs thus can be used in assay methods in place of DNA orRNA. Although assays are described herein utilizing DNA probes, it iswithin the scope of the routineer that PNAs or MAs can be substitutedfor RNA or DNA with appropriate changes if and as needed in assayreagents.

“Analyte,” as used herein, is the substance to be detected which may bepresent in the test sample. The analyte can be any substance for whichthere exists a naturally occurring specific binding member (such as, anantibody), or for which a specific binding member can be prepared. Thus,an analyte is a substance that can bind to one or more specific bindingmembers in an assay. “Analyte” also includes any antigenic substances,haptens, antibodies and combinations thereof. As a member of a specificbinding pair, the analyte can be detected by means of naturallyoccurring specific binding partners (pairs) such as the use of intrinsicfactor protein as a member of a specific binding pair for thedetermination of Vitamin B12, the use of folate-binding protein todetermine folic acid, or the use of a lectin as a member of a specificbinding pair for the determination of a carbohydrate. The analyte caninclude a protein, a polypeptide, an amino acid, a nucleotide target andthe like.

“Diseases of the prostate” or “prostate disease,” or “condition of theprostate,” as used herein, refer to any disease or condition of theprostate including, but not limited to, benign prostatic hyperplasia(BPH), prostatitis, prostatic intraepithelial neoplasia (PIN) andcancer.

“Prostate cancer,” as used herein, refers to any malignant disease ofthe prostate including, but not limited to, adenocarcinoma, small cellundifferentiated carcinoma and mucinous (colloid) cancer.

An “Expressed Sequence Tag” or “EST” refers to the partial sequence of acDNA insert which has been made by reverse transcription of mRNAextracted from a tissue followed by insertion into a vector.

A “transcript image” refers to a table or list giving the quantitativedistribution of ESTs in a library and represents the genes active in thetissue from which the library was made.

The present invention provides assays which utilize specific bindingmembers. A “specific binding member,” as used herein, is a member of aspecific binding pair. That is, two different molecules where one of themolecules, through chemical or physical means, specifically binds to thesecond molecule. Therefore, in addition to antigen and antibody specificbinding pairs of common immunoassays, other specific binding pairs caninclude biotin and avidin, carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors, and enzymes and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, antibodies and antibody fragments, both monoclonal andpolyclonal and complexes thereof, including those formed by recombinantDNA molecules.

The term “hapten,” as used herein, refers to a partial antigen ornon-protein binding member which is capable of binding to an antibody,but which is not capable of eliciting antibody formation unless coupledto a carrier protein.

A “capture reagent,” as used herein, refers to an unlabeled specificbinding member which is specific either for the analyte as in a sandwichassay, for the indicator reagent or analyte as in a competitive assay,or for an ancillary specific binding member, which itself is specificfor the analyte, as in an indirect assay. The capture reagent can bedirectly or indirectly bound to a solid phase material before theperformance of the assay or during the performance of the assay, therebyenabling the separation of immobilized complexes from the test sample.

“Specific binding member” as used herein means a member of a specificbinding pair. That is, two different molecules where one of themolecules through chemical or physical means specifically binds to thesecond molecule.

The “indicator reagent” comprises a “signal-generating compound”(“label”) which is capable of generating and generates a measurablesignal detectable by external means, conjugated (“attached”) to aspecific binding member. In addition to being an antibody member of aspecific binding pair, the indicator reagent also can be a member of anyspecific binding pair, including either hapten-anti-hapten systems suchas biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin,a complementary nucleotide sequence, an effector or a receptor molecule,an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme andthe like. An immunoreactive specific binding member can be an antibody,an antigen, or an antibody/antigen complex that is capable of bindingeither to the polypeptide of interest as in a sandwich assay, to thecapture reagent as in a competitive assay, or to the ancillary specificbinding member as in an indirect assay. When describing probes and probeassays, the term “reporter molecule” may be used. A reporter moleculecomprises a signal generating compound as described hereinaboveconjugated to a specific binding member of a specific binding pair, suchas carbazole or adamantane.

The various “signal-generating compounds” (labels) contemplated includechromagens, catalysts such as enzymes, luminescent compounds such asfluorescein and rhodamine, chemiluminescent compounds such asdioxetanes, acridiniums, phenanthridiniums and luminol, radioactiveelements and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase and the like.The selection of a particular label is not critical, but it must becapable of producing a signal either by itself or in conjunction withone or more additional substances.

“Solid phases” (“solid supports”) are known to those in the art andinclude the walls of wells of a reaction tray, test tubes, polystyrenebeads, magnetic or non-magnetic beads, nitrocellulose strips, membranes,microparticles such as latex particles, sheep (or other animal) redblood cells and Duracytes® (red blood cells “fixed” by pyruvic aldehydeand formaldehyde, available from Abbott Laboratories, Abbott Park, Ill.)and others. The “solid phase” is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and Duracytes® are all suitable examples. Suitable methods forimmobilizing peptides on solid phases include ionic, hydrophobic,covalent interactions and the like. A “solid phase,” as used herein,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. The solid phase can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid phase and which has the ability to immobilize the capturereagent through a specific binding reaction. The receptor moleculeenables the indirect binding of the capture reagent to a solid phasematerial before the performance of the assay or during the performanceof the assay. The solid phase thus can be a plastic, derivatizedplastic, magnetic or non-magnetic metal, glass or silicon surface of atest tube, microtiter well, sheet, bead, microparticle, chip, sheep (orother suitable animal's) red blood cells, Duracytes® and otherconfigurations known to those of ordinary skill in the art.

It is contemplated and within the scope of the present invention thatthe solid phase also can comprise any suitable porous material withsufficient porosity to allow access by detection antibodies and asuitable surface affinity to bind antigens. Microporous structuresgenerally are preferred, but materials with a gel structure in thehydrated state may be used as well. Such useful solid supports include,but are not limited to, nitrocellulose and nylon. It is contemplatedthat such porous solid supports described herein preferably are in theform of sheets of thickness from about 0.01 to 0.5 mm, preferably about0.1 mm. The pore size may vary within wide limits and preferably is fromabout 0.025 to 15 microns, especially from about 0.15 to 15 microns. Thesurface of such supports may be activated by chemical processes whichcause covalent linkage of the antigen or antibody to the support. Theirreversible binding of the antigen or antibody is obtained, however, ingeneral, by adsorption on the porous material by poorly understoodhydrophobic forces. Other suitable solid supports are known in the art.

Reagents

The present invention provides reagents such as polynucleotide sequencesderived from a prostate tissue of interest and designated as PS190,polypeptides encoded thereby and antibodies specific for thesepolypeptides. The present invention also provides reagents such asoligonucleotide fragments derived from the disclosed polynucleotides andnucleic acid sequences complementary to these polynucleotides. Thepolynucleotides, polypeptides, or antibodies of the present inventionmay be used to provide information leading to the detecting, diagnosing,staging, monitoring, prognosticating, preventing or treating of, ordetermining the predisposition to, diseases and conditions of theprostate such as cancer. The sequences disclosed herein represent uniquepolynucleotides which can be used in assays or for producing a specificprofile of gene transcription activity. Such assays are disclosed inEuropean Patent Number 0373203B1 and International Publication No. WO95/11995, which are hereby incorporated by reference.

Selected PS190-derived polynucleotides can be used in the methodsdescribed herein for the detection of normal or altered gene expression.Such methods may employ PS190 polynucleotides or oligonucleotides,fragments or derivatives thereof, or nucleic acid sequencescomplementary thereto.

The polynucleotides disclosed herein, their complementary sequences, orfragments of either, can be used in assays to detect, amplify orquantify genes, nucleic acids, cDNAs or mRNAs relating to prostatetissue disease and conditions associated therewith. They also can beused to identify an entire or partial coding region of a PS190polypeptide. They further can be provided in individual containers inthe form of a kit for assays, or provided as individual compositions. Ifprovided in a kit for assays, other suitable reagents such as buffers,conjugates and the like may be included.

The polynucleotide may be in the form of RNA or DNA. Polynucleotides inthe form of DNA, cDNA, genomic DNA, nucleic acid analogs and syntheticDNA are within the scope of the present invention. The DNA may bedouble-stranded or single-stranded, and if single stranded, may be thecoding (sense) strand or non-coding (anti-sense) strand. The codingsequence which encodes the polypeptide may be identical to the codingsequence provided herein or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same polypeptide as the DNA provided herein.

This polynucleotide may include only the coding sequence for thepolypeptide, or the coding sequence for the polypeptide and anadditional coding sequence such as a leader or secretory sequence or aproprotein sequence, or the coding sequence for the polypeptide (andoptionally an additional coding sequence) and non-coding sequence, suchas a non-coding sequence 5′ and/or 3′ of the coding sequence for thepolypeptide.

In addition, the invention includes variant polynucleotides containingmodifications such as polynucleotide deletions, substitutions oradditions; and any polypeptide modification resulting from the variantpolynucleotide sequence. A polynucleotide of the present invention alsomay have a coding sequence which is a naturally occurring allelicvariant of the coding sequence provided herein.

In addition, the coding sequence for the polypeptide may be fused in thesame reading frame to a polynucleotide sequence which aids in expressionand secretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the polypeptide. The polynucleotides may alsoencode for a proprotein which is the protein plus additional 5′ aminoacid residues. A protein having a prosequence is a proprotein and may,in some cases, be an inactive form of the protein. Once the prosequenceis cleaved, an active protein remains. Thus, the polynucleotide of thepresent invention may encode for a protein, or for a protein having aprosequence, or for a protein having both a presequence (leadersequence) and a prosequence.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the polypeptide fused to the marker in thecase of a bacterial host, or, for example, the marker sequence may be ahemagglutinin (HA) tag when a mammalian host, e.g. a COS-7 cell line, isused. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein. See, for example, I. Wilson, et al., Cell 37:767(1984).

It is contemplated that polynucleotides will be considered to hybridizeto the sequences provided herein if there is at least 50%, preferably atleast 70%, and more preferably at least 90% identity between thepolynucleotide and the sequence.

The present invention also provides an antibody produced by using apurified PS190 polypeptide of which at least a portion of thepolypeptide is encoded by a PS190 polynucleotide selected from thepolynucleotides provided herein. These antibodies may be used in themethods provided herein for the detection of PS190 antigen in testsamples. The presence of PS190 antigen in the test samples is indicativeof the presence of a prostate disease or condition. The antibody alsomay be used for therapeutic purposes, for example, in neutralizing theactivity of PS190 polypeptide in conditions associated with altered orabnormal expression.

The present invention further relates to a PS190 polypeptide which hasthe deduced amino acid sequence as provided herein, as well asfragments, analogs and derivatives of such polypeptide. The polypeptideof the present invention may be a recombinant polypeptide, a naturalpurified polypeptide or a synthetic polypeptide. The fragment,derivative or analog of the PS190 polypeptide may be one in which one ormore of the amino acid residues is substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or it may be one in which one or more ofthe amino acid residues includes a substituent group; or it may be onein which the polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol); or it may be one in which the additional aminoacids are fused to the polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of thepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are within the scope of the present invention. The polypeptidesand polynucleotides of the present invention are provided preferably inan isolated form and preferably purified.

Thus, a polypeptide of the present invention may have an amino acidsequence that is identical to that of the naturally occurringpolypeptide or that is different by minor variations due to one or moreamino acid substitutions. The variation may be a “conservative change”typically in the range of about 1 to 5 amino acids, wherein thesubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine or threonine with serine.In contrast, variations may include nonconservative changes, e.g.,replacement of a glycine with a tryptophan. Similar minor variations mayalso include amino acid deletions or insertions, or both. Guidance indetermining which and how many amino acid residues may be substituted,inserted or deleted without changing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software (DNASTAR Inc., Madison Wis.).

Probes constructed according to the polynucleotide sequences of thepresent invention can be used in various assay methods to providevarious types of analysis. For example, such probes can be used influorescent in situ hybridization (FISH) technology to performchromosomal analysis, and used to identify cancer-specific structuralalterations in the chromosomes, such as deletions or translocations thatare visible from chromosome spreads or detectable using PCR-generatedand/or allele specific oligonucleotides probes, allele specificamplification or by direct sequencing. Probes also can be labeled withradioisotopes, directly- or indirectly- detectable haptens, orfluorescent molecules, and utilized for in situ hybridization studies toevaluate the mRNA expression of the gene comprising the polynucleotidein tissue specimens or cells.

This invention also provides teachings as to the production of thepolynucleotides and polypeptides provided herein.

Probe Assays

The sequences provided herein may be used to produce probes which can beused in assays for the detection of nucleic acids in test samples. Theprobes may be designed from conserved nucleotide regions of thepolynucleotides of interest or from non-conserved nucleotide regions ofthe polynucleotide of interest. The design of such probes foroptimization in assays is within the skill of the routineer. Generally,nucleic acid probes are developed from non-conserved or unique regionswhen maximum specificity is desired, and nucleic acid probes aredeveloped from conserved regions when assaying for nucleotide regionsthat are closely related to, for example, different members of amulti-gene family or in related species like mouse and man.

The polymerase chain reaction (PCR) is a technique for amplifying adesired nucleic acid sequence (target) contained in a nucleic acid ormixture thereof. In PCR, a pair of primers are employed in excess tohybridize to the complementary strands of the target nucleic acid. Theprimers are each extended by a polymerase using the target nucleic acidas a template. The extension products become target sequencesthemselves, following dissociation from the original target strand. Newprimers then are hybridized and extended by a polymerase, and the cycleis repeated to geometrically increase the number of target sequencemolecules. PCR is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202,which are incorporated herein by reference.

The Ligase Chain Reaction (LCR) is an alternate method for nucleic acidamplification. In LCR, probe pairs are used which include two primary(first and second) and two secondary (third and fourth) probes, all ofwhich are employed in molar excess to target. The first probe hybridizesto a first segment of the target strand, and the second probe hybridizesto a second segment of the target strand, the first and second segmentsbeing contiguous so that the primary probes abut one another in 5′phosphate-3′ hydroxyl relationship, and so that a ligase can covalentlyfuse or ligate the two probes into a fused product. In addition, a third(secondary) probe can hybridize to a portion of the first probe and afourth (secondary) probe can hybridize to a portion of the second probein a similar abutting fashion. Of course, if the target is initiallydouble stranded, the secondary probes also will hybridize to the targetcomplement in the first instance. Once the ligated strand of primaryprobes is separated from the target strand, it will hybridize with thethird and fourth probes which can be ligated to form a complementary,secondary ligated product. It is important to realize that the ligatedproducts are functionally equivalent to either the target or itscomplement. By repeated cycles of hybridization and ligation,amplification of the target sequence is achieved. This technique isdescribed more completely in EP-A-320 308 to K. Backman published Jun.16, 1989 and EP-A-439 182 to K. Backman et al., published Jul. 31, 1991,both of which are incorporated herein by reference.

For amplification of mRNAs, it is within the scope of the presentinvention to reverse transcribe mRNA into cDNA followed by polymerasechain reaction (RT-PCR); or, to use a single enzyme for both steps asdescribed in U.S. Pat. No. 5,322,770, which is incorporated herein byreference; or reverse transcribe mRNA into cDNA followed by asymmetricgap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall etal., PCR Methods and Applications 4: 80-84 (1994), which also isincorporated herein by reference.

Other known amplification methods which can be utilized herein includebut are not limited to the so-called “NASBA” or “3SR” techniquedescribed by J. C. Guatelli et al., PNAS USA 87:1874-1878 (1990) andalso described by J. Compton, Nature 350 (No. 6313):91-92 (1991); Q-betaamplification as described in published European Patent Application(EPA) No. 4544610; strand displacement amplification (as described in G.T. Walker et al., Clin. Chem. 42:9-13 [1996]) and European PatentApplication No. 684315; and target mediated amplification, as describedin International Publication No. WO 93/22461.

Detection of PS190 may be accomplished using any suitable detectionmethod, including those detection methods which are currently well knownin the art, as well as detection strategies which may evolve later.Examples of the foregoing presently known detection methods are herebyincorporated herein by reference. See, for example, Caskey et al., U.S.Pat. No. 5,582,989, Gelfand et al., U.S. Pat. No. 5,210,015. Examples ofsuch detection methods include target amplification methods as well assignal amplification technologies. An example of presently knowndetection methods would include the nucleic acid amplificationtechnologies referred to as PCR, LCR, NASBA, SDA, RCR and TMA. See, forexample, Caskey et al., U.S. Pat. No. 5,582,989, Gelfand et al., U.S.Pat. No. 5,210,015. All of the foregoing are hereby incorporated byreference. Detection may also be accomplished using signal amplificationsuch as that disclosed in Snitman et al., U.S. Pat. No. 5,273,882. Whilethe amplification of target or signal is preferred at present, it iscontemplated and within the scope of the present invention thatultrasensitive detection methods which do not require amplification canbe utilized herein.

Detection, both amplified and non-amplified, may be (combined) carriedout using a variety of heterogeneous and homogeneous detection formats.Examples of heterogeneous detection formats are disclosed in Snitman etal., U.S. Pat. No. 5,273,882, Albarella et al. in EP-84114441.9, Urdeaet al., U.S. Pat. No. 5,124,246, Ullman et al. U.S. Pat. No. 5,185,243and Kourilsky et al., U.S. Pat. No. 4,581,333. All of the foregoing arehereby incorporated by reference. Examples of homogeneous detectionformats are disclosed in, Caskey et al., U.S. Pat. No. 5,582,989,Gelfand et al., U.S. Pat. No. 5,210,015, which are incorporated hereinby reference. Also contemplated and within the scope of the presentinvention is the use of multiple probes in the hybridization assay,which use improves sensitivity and amplification of the PS190 signal.See, for example, Caskey et al., U.S. Pat. No. 5,582,989, Gelfand etal., U.S. Pat. No. 5,210,015, which are incorporated herein byreference.

In one embodiment, the present invention generally comprises the stepsof contacting a test sample suspected of containing a targetpolynucleotide sequence with amplification reaction reagents comprisingan amplification primer, and a detection probe that can hybridize withan internal region of the amplicon sequences. Probes and primersemployed according to the method provided herein are labeled withcapture and detection labels, wherein probes are labeled with one typeof label and primers are labeled with another type of label.Additionally, the primers and probes are selected such that the probesequence has a lower melt temperature than the primer sequences. Theamplification reagents, detection reagents and test sample are placedunder amplification conditions whereby, in the presence of targetsequence, copies of the target sequence (an amplicon) are produced. Inthe usual case, the amplicon is double stranded because primers areprovided to amplify a target sequence and its complementary strand. Thedouble stranded amplicon then is thermally denatured to produce singlestranded amplicon members. Upon formation of the single strandedamplicon members, the mixture is cooled to allow the formation ofcomplexes between the probes and single stranded amplicon members.

As the single stranded amplicon sequences and probe sequences arecooled, the probe sequences preferentially bind the single strandedamplicon members. This finding is counterintuitive given that the probesequences generally are selected to be shorter than the primer sequencesand therefore have a lower melt temperature than the primers.Accordingly, the melt temperature of the amplicon produced by theprimers should also have a higher melt temperature than the probes.Thus, as the mixture cools, the re-formation of the double strandedamplicon would be expected. As previously stated, however, this is notthe case. The probes are found to preferentially bind the singlestranded amplicon members. Moreover, this preference of probe/singlestranded amplicon binding exists even when the primer sequences areadded in excess of the probes.

After the probe/single stranded amplicon member hybrids are formed, theyare detected. Standard heterogeneous assay formats are suitable fordetecting the hybrids using the detection labels and capture labelspresent on the primers and probes. The hybrids can be bound to a solidphase reagent by virtue of the capture label and detected by virtue ofthe detection label. In cases where the detection label is directlydetectable, the presence of the hybrids on the solid phase can bedetected by causing the label to produce a detectable signal, ifnecessary, and detecting the signal. In cases where the label is notdirectly detectable, the captured hybrids can be contacted with aconjugate, which generally comprises a binding member attached to adirectly detectable label. The conjugate becomes bound to the complexesand the conjugate's presence on the complexes can be detected with thedirectly detectable label. Thus, the presence of the hybrids on thesolid phase reagent can be determined. Those skilled in the art willrecognize that wash steps may be employed to wash away unhybridizedamplicon or probe as well as unbound conjugate.

Although the target sequence is described as single stranded, it also iscontemplated to include the case where the target sequence is actuallydouble stranded but is merely separated from its complement prior tohybridization with the amplification primer sequences. In the case wherePCR is employed in this method, the ends of the target sequences areusually known. In cases where LCR or a modification thereof is employedin the preferred method, the entire target sequence is usually known.Typically, the target sequence is a nucleic acid sequence such as, forexample, RNA or DNA.

The method provided herein can be used in well-known amplificationreactions that include thermal cycle reaction mixtures, particularly inPCR and gap LCR (GLCR). Amplification reactions typically employ primersto repeatedly generate copies of a target nucleic acid sequence, whichtarget sequence is usually a small region of a much larger nucleic acidsequence. Primers are themselves nucleic acid sequences that arecomplementary to regions of a target sequence. Under amplificationconditions, these primers hybridize or bind to the complementary regionsof the target sequence. Copies of the target sequence typically aregenerated by the process of primer extension and/or ligation whichutilizes enzymes with polymerase or ligase activity, separately or incombination, to add nucleotides to the hybridized primers and/or ligateadjacent probe pairs. The nucleotides that are added to the primers orprobes, as monomers or preformed oligomers, are also complementary tothe target sequence. Once the primers or probes have been sufficientlyextended and/or ligated, they are separated from the target sequence,for example, by heating the reaction mixture to a “melt temperature”which is one in which complementary nucleic acid strands dissociate.Thus, a sequence complementary to the target sequence is formed.

A new amplification cycle then can take place to further amplify thenumber of target sequences by separating any double stranded sequences,allowing primers or probes to hybridize to their respective targets,extending and/or ligating the hybridized primers or probes andre-separating. The complementary sequences that are generated byamplification cycles can serve as templates for primer extension orfilling the gap of two probes to further amplify the number of targetsequences. Typically, a reaction mixture is cycled between 20 and 100times, more typically, a reaction mixture is cycled between 25 and 50times. The numbers of cycles can be determined by the routineer. In thismanner, multiple copies of the target sequence and its complementarysequence are produced. Thus, primers initiate amplification of thetarget sequence when it is present under amplification conditions.

Generally, two primers which are complementary to a portion of a targetstrand and its complement are employed in PCR. For LCR, four probes, twoof which are complementary to a target sequence and two of which aresimilarly complementary to the target's complement, are generallyemployed. In addition to the primer sets and enzymes previouslymentioned, a nucleic acid amplification reaction mixture may alsocomprise other reagents which are well known and include but are notlimited to: enzyme cofactors such as manganese; magnesium; salts;nicotinamide adenine dinucleotide (NAD); and deoxynucleotidetriphosphates (dNTPs) such as, for example, deoxyadenine triphosphate,deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythyminetriphosphate.

While the amplification primers initiate amplification of the targetsequence, the detection (or hybridization) probe is not involved inamplification. Detection probes are generally nucleic acid sequences oruncharged nucleic acid analogs such as, for example, peptide nucleicacids which are disclosed in International Publication No. WO 92/20702;morpholino analogs which are described in U.S. Pat. Nos. 5,185,444,5,034,506 and 5,142,047; and the like. Depending upon the type of labelcarried by the probe, the probe is employed to capture or detect theamplicon generated by the amplification reaction. The probe is notinvolved in amplification of the target sequence and therefore may haveto be rendered “non-extendible” in that additional dNTPs cannot be addedto the probe. In and of themselves, analogs usually are non-extendibleand nucleic acid probes can be rendered non-extendible by modifying the3′ end of the probe such that the hydroxyl group is no longer capable ofparticipating in elongation. For example, the 3′ end of the probe can befunctionalized with the capture or detection label to thereby consume orotherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl groupsimply can be cleaved, replaced or modified. U.S. patent applicationSer. No. 07/049,061 filed Apr. 19, 1993 and incorporated herein byreference describes modifications which can be used to render a probenon-extendible.

The ratio of primers to probes is not important. Thus, either the probesor primers can be added to the reaction mixture in excess whereby theconcentration of one would be greater than the concentration of theother. Alternatively, primers and probes can be employed in equivalentconcentrations. Preferably, however, the primers are added to thereaction mixture in excess of the probes. Thus, primer to probe ratiosof, for example, 5:1 and 20:1 are preferred.

While the length of the primers and probes can vary, the probe sequencesare selected such that they have a lower melt temperature than theprimer sequences. Hence, the primer sequences are generally longer thanthe probe sequences. Typically, the primer sequences are in the range ofbetween 20 and 50 nucleotides long, more typically in the range ofbetween 20 and 30 nucleotides long. The typical probe is in the range ofbetween 10 and 25 nucleotides long.

Various methods for synthesizing primers and probes are well known inthe art. Similarly, methods for attaching labels to primers or probesare also well known in the art. For example, it is a matter of routineto synthesize desired nucleic acid primers or probes using conventionalnucleotide phosphoramidite chemistry and instruments available fromApplied Biosystems, Inc., (Foster City, Calif.), DuPont (Wilmington,Del.), or Milligen (Bedford Mass.). Many methods have been described forlabeling oligonucleotides such as the primers or probes of the presentinvention. Enzo Biochemical (New York, N.Y.) and Clontech (Palo Alto,Calif.) both have described and commercialized probe labelingtechniques. For example, a primary amine can be attached to a 3′ oligoterminus using 3′-Amine-ON CPG™ (Clontech, Palo Alto, Calif.).Similarly, a primary amine can be attached to a 5′ oligo terminus usingAminomodifier II® (Clontech). The amines can be reacted to varioushaptens using conventional activation and linking chemistries. Inaddition, copending applications U.S. Ser. Nos. 625,566, filed Dec. 11,1990 and 630,908, filed Dec. 20, 1990, which are each incorporatedherein by reference, teach methods for labeling probes at their 5′ and3′ termini, respectively. International Publication Nos WO 92/10505,published Jun. 25, 1992, and WO 92/11388, published Jul. 9, 1992, teachmethods for labeling probes at their 5′ and 3′ ends, respectively.According to one known method for labeling an oligonucleotide, alabel-phosphoramidite reagent is prepared and used to add the label tothe oligonucleotide during its synthesis. See, for example, N.T. Thuonget al., Tet. Letters 29(46):5905-5908 (1988); or J. S. Cohen et al.,published U.S. patent application Ser. No. 07/246,688 (NTIS ORDER No.PAT-APPL-7-246,688) (1989). Preferably, probes are labeled at their 3′and 5′ ends.

A capture label is attached to the primers or probes and can be aspecific binding member which forms a binding pair with the solid phasereagent's specific binding member. It will be understood that the primeror probe itself may serve as the capture label. For example, in the casewhere a solid phase reagent's binding member is a nucleic acid sequence,it may be selected such that it binds a complementary portion of theprimer or probe to thereby immobilize the primer or probe to the solidphase. In cases where the probe itself serves as the binding member,those skilled in the art will recognize that the probe will contain asequence or “tail” that is not complementary to the single strandedamplicon members. In the case where the primer itself serves as thecapture label, at least a portion of the primer will be free tohybridize with a nucleic acid on a solid phase because the probe isselected such that it is not fully complementary to the primer sequence.

Generally, probe/single stranded amplicon member complexes can bedetected using techniques commonly employed to perform heterogeneousimmunoassays. Preferably, in this embodiment, detection is performedaccording to the protocols used by the commercially available AbbottLCx® instrumentation (Abbott Laboratories, Abbott Park, Ill.).

The primers and probes disclosed herein are useful in typical PCRassays, wherein the test sample is contacted with a pair of primers,amplification is performed, the hybridization probe is added, anddetection is performed.

Another method provided by the present invention comprises contacting atest sample with a plurality of polynucleotides, wherein at least onepolynucleotide is a PS190 molecule as described herein, hybridizing thetest sample with the plurality of polynucleotides and detectinghybridization complexes. Hybridization complexes are identified andquantitated to compile a profile which is indicative of prostate tissuedisease, such as prostate cancer. Expressed RNA sequences may further bedetected by reverse transcription and amplification of the DNA productby procedures well-known in the art, including polymerase chain reaction(PCR).

Drug Screening and Gene Therapy.

The present invention also encompasses the use of gene therapy methodsfor the introduction of anti-sense PS190 derived molecules, such aspolynucleotides or oligonucleotides of the present invention, intopatients with conditions associated with abnormal expression ofpolynucleotides related to a prostate tissue disease or condition,especially prostate cancer. These molecules, including antisense RNA andDNA fragments and ribozymes, are designed to inhibit the translation ofPS190-mRNA, and may be used therapeutically in the treatment ofconditions associated with altered or abnormal expression of PS190polynucleotide.

Alternatively, the oligonucleotides described above can be delivered tocells by procedures known in the art such that the anti-sense RNA or DNAmay be expressed in vivo to inhibit production of a PS190 polypeptide inthe manner described above. Antisense constructs to a PS190polynucleotide, therefore, reverse the action of PS190 transcripts andmay be used for treating prostate tissue disease conditions, such asprostate cancer. These antisense constructs may also be used to treattumor metastases.

The present invention also provides a method of screening a plurality ofcompounds for specific binding to PS190 polypeptide(s), or any fragmentthereof, to identify at least one compound which specifically binds thePS190 polypeptide. Such a method comprises the steps of providing atleast one compound; combining the PS190 polypeptide with each compoundunder suitable conditions for a time sufficient to allow binding; anddetecting the PS190 polypeptide binding to each compound.

The polypeptide or peptide fragment employed in such a test may eitherbe free in solution, affixed to a solid support, borne on a cell surfaceor located intracellularly. One method of drug screening utilizeseukaryotic or prokaryotic host cells which are stably transfected withrecombinant nucleic acids which can express the polypeptide or peptidefragment. Drugs may be screened against such transfected cells incompetitive binding assays. For example, the formation of complexesbetween a polypeptide and the agent being tested can be measured ineither viable or fixed cells.

The present invention thus provides methods of screening for drugs orany other agent which can be used to treat diseases associated withPS190. These methods comprise contacting the drug with a polypeptide orfragment thereof and assaying for either the presence of a complexbetween the agent and the polypeptide, or for the presence of a complexbetween the polypeptide and the cell. In competitive binding assays, thepolypeptide typically is labeled. After suitable incubation, free (oruncomplexed) polypeptide or fragment thereof is separated from thatpresent in bound form, and the amount of free or uncomplexed label isused as a measure of the ability of the particular drug to bind to thepolypeptide or to interfere with the polypeptide/cell complex.

The present invention also encompasses the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingpolypeptide specifically compete with a test drug for binding to thepolypeptide or fragment thereof. In this manner, the antibodies can beused to detect the presence of any polypeptide in the test sample whichshares one or more antigenic determinants with a PS190 polypeptide asprovided herein.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to at least onepolypeptide of PS190 disclosed herein. Briefly, large numbers ofdifferent small peptide test compounds are synthesized on a solid phase,such as plastic pins or some other surface. The peptide test compoundsare reacted with polypeptide and washed. Polypeptide thus bound to thesolid phase is detected by methods well-known in the art. Purifiedpolypeptide can also be coated directly onto plates for use in the drugscreening techniques described herein. In addition, non-neutralizingantibodies can be used to capture the polypeptide and immobilize it onthe solid support. See, for example, EP 84/03564, published on Sep. 13,1984, which is incorporated herein by reference.

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of the small moleculesincluding agonists, antagonists, or inhibitors with which they interact.Such structural analogs can be used to design drugs which are moreactive or stable forms of the polypeptide or which enhance or interferewith the function of a polypeptide in vivo. J. Hodgson, Bio/Technology9:19-21 (1991), incorporated herein by reference.

For example, in one approach, the three-dimensional structure of apolypeptide, or of a polypeptide-inhibitor complex, is determined byx-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous polypeptide-likemolecules or to identify efficient inhibitors

Useful examples of rational drug design may include molecules which haveimproved activity or stability as shown by S. Braxton et al.,Biochemistry 31:7796-7801 (1992), or which act as inhibitors, agonists,or antagonists of native peptides as shown by S. B. P. Athauda et al.,J. Biochem. (Tokyo) 113 (6):742-746 (1993), incorporated herein byreference.

It also is possible to isolate a target-specific antibody selected by anassay as described hereinabove, and then to determine its crystalstructure. In principle this approach yields a pharmacophore upon whichsubsequent drug design can be based. It further is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (“anti-ids”) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-id is an analog of the original receptor. The anti-id then can beused to identify and isolate peptides from banks of chemically orbiologically produced peptides. The isolated peptides then can act asthe pharmacophore (that is, a prototype pharmaceutical drug).

A sufficient amount of a recombinant polypeptide of the presentinvention may be made available to perform analytical studies such asX-ray crystallography. In addition, knowledge of the polypeptide aminoacid sequence which is derivable from the nucleic acid sequence providedherein will provide guidance to those employing computer modelingtechniques in place of, or in addition to, x-ray crystallography.

Antibodies specific to a PS190 polypeptide (e.g., anti-PS190 antibodies)further may be used to inhibit the biological action of the polypeptideby binding to the polypeptide. In this manner, the antibodies may beused in therapy, for example, to treat prostate tissue diseasesincluding prostate cancer and its metastases.

Further, such antibodies can detect the presence or absence of a PS190polypeptide in a test sample and, therefore, are useful as diagnosticmarkers for the diagnosis of a prostate tissue disease or condition,especially prostate cancer. Such antibodies may also function as adiagnostic marker for prostate tissue disease conditions, such asprostate cancer. The present invention also is directed to antagonistsand inhibitors of the polypeptides of the present invention. Theantagonists and inhibitors are those which inhibit or eliminate thefunction of the polypeptide. Thus, for example, an antagonist may bindto a polypeptide of the present invention and inhibit or eliminate itsfunction. The antagonist, for example, could be an antibody against thepolypeptide which eliminates the activity of a PS190 polypeptide bybinding a PS190 polypeptide, or in some cases the antagonist may be anoligonucleotide. Examples of small molecule inhibitors include, but arenot limited to, small peptides or peptide-like molecules.

The antagonists and inhibitors may be employed as a composition with apharmaceutically acceptable carrier including, but not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol andcombinations thereof. Administration of PS190 polypeptide inhibitors ispreferably systemic. The present invention also provides an antibodywhich inhibits the action of such a polypeptide.

Antisense technology can be used to reduce gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes for thepolypeptide of the present invention, is used to design an antisense RNAoligonucleotide of from 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription, thereby preventing transcription and theproduction of the PS190 polypeptide. For triple helix, see, for example,Lee et al., Nuc. Acids Res. 6:3073 (1979); Cooney et al., Science241:456 (1988); and Dervan et al., Science 251:1360 (1991) The antisenseRNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of a mRNA molecule into the PS190 polypeptide. Forantisense, see, for example, Okano, J. Neurochem. 56:560 (1991); and“Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,” CRCPress, Boca Raton, Fla. (1988). Antisense oligonucleotides act withgreater efficacy when modified to contain artificial intemucleotidelinkages which render the molecule resistant to nucleolytic cleavage.Such artificial intemucleotide linkages include, but are not limited to,methylphosphonate, phosphorothiolate and phosphoroamydate intemucleotidelinkages.

Recombinant Technology

The present invention provides host cells and expression vectorscomprising PS190 polynucleotides of the present invention and methodsfor the production of the polypeptide(s) they encode. Such methodscomprise culturing the host cells under conditions suitable for theexpression of the PS190 polynucleotide and recovering the PS190polypeptide from the cell culture.

The present invention also provides vectors which include PS190polynucleotides of the present invention, host cells which aregenetically engineered with vectors of the present invention and theproduction of polypeptides of the present invention by recombinanttechniques.

Host cells are genetically engineered (transfected, transduced ortransformed) with the vectors of this invention which may be cloningvectors or expression vectors. The vector may be in the form of aplasmid, a viral particle, a phage, etc. The engineered host cells canbe cultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transfected cells, or amplifying PS190gene(s). The culture conditions, such as temperature, pH and the like,are those previously used with the host cell selected for expression,and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, thepolynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virusand pseudorabies. However, any other plasmid or vector may be used solong as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted intoappropriate restriction endonuclease sites by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art. The DNA sequence in the expression vector isoperatively linked to an appropriate expression control sequence(s)(promoter) to direct mRNA synthesis. Representative examples of suchpromoters include, but are not limited to, the LTR or the SV40 promoter,the E. coli lac or trp, the phage lambda P sub L promoter and otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. The expression vector also contains aribosome binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression. In addition, the expression vectors preferablycontain a gene to provide a phenotypic trait for selection oftransfected host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transfect an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium; Streptomyces sp; fungal cells, such as yeast; insect cellssuch as Drosophila and Sf9; animal cells such as CHO, COS or Bowesmelanoma; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings provided herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available. The following vectorsare provided by way of example. Bacterial: pINCY (Incyte PharmaceuticalsInc., Palo Alto, Calif.), pSPORT1 (Life Technologies, Gaithersburg,Md.), pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript, psiX174,pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic:pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL(Pharmacia). However, any other plasmid or vector may be used as long asit is replicable and viable in the host.

Plasmid pINCY is generally identical to the plasmid pSPORT1 (availablefrom Life Technologies, Gaithersburg, Md.) with the exception that ithas two modifications in the polylinker (multiple cloning site). Thesemodifications are (1) it lacks a HindIII restriction site and (2) itsEcoRI restriction site lies at a different location. pINCY is createdfrom pSPORT1 by cleaving pSPORT1 with both HindIII and EcoRI andreplacing the excised fragment of the polylinker with synthetic DNAfragments (SEQUENCE ID NO 5 and SEQUENCE ID NO 6). This replacement maybe made in any manner known to those of ordinary skill in the art. Forexample, the two nucleotide sequences, SEQUENCE ID NO 5 and SEQUENCE IDNO 6, may be generated synthetically with 5′ terminal phosphates, mixedtogether, and then ligated under standard conditions for performingstaggered end ligations into the pSPORT1 plasmid cut with HindIII andEcoRI. Suitable host cells (such as E. coli DH5∝ cells) then aretransfected with the ligated DNA and recombinant clones are selected forampicillin resistance. Plasmid DNA then is prepared from individualclones and subjected to restriction enzyme analysis or DNA sequencing inorder to confirm the presence of insert sequences in the properorientation. Other cloning strategies known to the ordinary artisan alsomay be employed.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacd, lacZ, T3, SP6, T7, gpt, lambda P subR, P sub L and trp. Eukaryotic promoters include cytomegalovirus (CMV)immediate early, herpes simplex virus (HSV) thymidine kinase, early andlate SV40, LTRs from retroviruses and mouse metallothionein-I. Selectionof the appropriate vector and promoter is well within the level ofordinary skill in the art.

In a further embodiment, the present invention provides host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (L. Davis et al., “Basic Methods inMolecular Biology,” 2nd edition, Appleton and Lang, ParamountPublishing, East Norwalk, Conn. (1994)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Recombinant proteins can be expressed in mammalian cells, yeast,bacteria, or other cells, under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor, N.Y., 1989), which is hereby incorporated by reference.

Transcription of a DNA encoding the polypeptide(s) of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication originand adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transfection of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransfection include E. coli, Bacillus subtilis, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, although, others may also be employed as a routinematter of choice.

Useful expression vectors for bacterial use comprise a selectable markerand bacterial origin of replication derived from plasmids comprisinggenetic elements of the well-known cloning vector pBR322 (ATCC 37017).Other vectors include but are not limited to PKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.).These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transfection of a suitable host and growth of the host to anappropriate cell density, the selected promoter is derepressed byappropriate means (e.g., temperature shift or chemical induction), andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents; such methods arewell-known to the ordinary artisan.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, such as the C127, HEK-293, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer and also any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements. Representative, useful vectors include pRc/CMV andpcDNA3 (available from Invitrogen, San Diego, Calif.).

PS190 polypeptides are recovered and purified from recombinant cellcultures by known methods including affinity chromatography, ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, hydroxyapatite chromatography or lectinchromatography. It is preferred to have low concentrations(approximately 0.1-5 mM) of calcium ion present during purification(Price et al., J. Biol. Chem. 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of thepolypeptide. Finally, high performance liquid chromatography (HPLC) canbe employed for final purification steps.

Thus, polypeptides of the present invention may be naturally purifiedproducts expressed from a high expressing cell line, or a product ofchemical synthetic procedures, or produced by recombinant techniquesfrom a prokaryotic or eukaryotic host (for example, by bacterial, yeast,higher plant, insect and mammalian cells in culture). Depending upon thehost employed in a recombinant production procedure, the polypeptides ofthe present invention may be glycosylated with mammalian or othereukaryotic carbohydrates or may be non-glycosylated. The polypeptides ofthe invention may also include an initial methionine amino acid residue.

The starting plasmids can be constructed from available plasmids inaccord with published, known procedures. In addition, equivalentplasmids to those described are known in the art and will be apparent tothe ordinarily skilled artisan.

The following is the general procedure for the isolation and analysis ofcDNA clones. In a particular embodiment disclosed herein, mRNA wasisolated from prostate tissue and used to generate the cDNA library.Prostate tissue was obtained from patients by surgical resection and wasclassified as tumor or non-tumor tissue by a pathologist.

The cDNA inserts from random isolates of the prostate tissue librarieswere sequenced in part, analyzed in detail as set forth in the Examplesand are disclosed in the Sequence Listing as SEQUENCE ID NO 1, SEQUENCEID NO 2, and SEQUENCE ID NO 3. The consensus sequence of these insertsis presented as SEQUENCE ID NO 4. These polynucleotides may contain anentire open reading frame with or without associated regulatorysequences for a particular gene, or they may encode only a portion ofthe gene of interest. This is attributed to the fact that many genes areseveral hundred and sometimes several thousand, bases in length and,with current technology, cannot be cloned in their entirety because ofvector limitations, incomplete reverse transcription of the firststrand, or incomplete replication of the second strand. Contiguous,secondary clones containing additional nucleotide sequences may beobtained using a variety of methods known to those of skill in the art.

Methods for DNA sequencing are well known in the art. Conventionalenzymatic methods employ DNA polymerase, Klenow fragment, Sequenase (USBiochemical Corp, Cleveland, Ohio) or Taq polymerase to extend DNAchains from an oligonucleotide primer annealed to the DNA template ofinterest. Methods have been developed for the use of bothsingle-stranded and double-stranded templates. The chain terminationreaction products may be electrophoresed on urea/polyacrylamide gels anddetected either by autoradiography (for radionucleotide labeledprecursors) or by fluorescence (for fluorescent-labeled precursors).Recent improvements in mechanized reaction preparation, sequencing andanalysis using the fluorescent detection method have permitted expansionin the number of sequences that can be determined per day using machinessuch as the Applied Biosystems 377 DNA Sequencers (Applied Biosystems,Foster City, Calif.).

The reading frame of the nucleotide sequence can be ascertained byseveral types of analyses. First, reading frames contained within thecoding sequence can be analyzed for the presence of start codon ATG andstop codons TGA, TAA or TAG. Typically, one reading frame will continuethroughout the major portion of a cDNA sequence while other readingframes tend to contain numerous stop codons. In such cases, readingframe determination is straightforward. In other more difficult cases,further analysis is required.

Algorithms have been created to analyze the occurrence of individualnucleotide bases at each putative codon triplet. See, for example J. W.Fickett, Nuc Acids Res 10:5303 (1982). Coding DNA for particularorganisms (bacteria, plants and animals) tends to contain certainnucleotides within certain triplet periodicities, such as a significantpreference for pyrimidines in the third codon position. Thesepreferences have been incorporated into widely available software whichcan be used to determine coding potential (and frame) of a given stretchof DNA. The algorithm-derived information combined with start/stop codoninformation can be used to determine proper frame with a high degree ofcertainty. This, in turn, readily permits cloning of the sequence in thecorrect reading frame into appropriate expression vectors.

The nucleic acid sequences disclosed herein may be joined to a varietyof other polynucleotide sequences and vectors of interest by means ofwell-established recombinant DNA techniques. See J. Sambrook et al.,supra. Vectors of interest include cloning vectors, such as plasmids,cosmids, phage derivatives, phagemids, as well as sequencing,replication and expression vectors, and the like. In general, suchvectors contain an origin of replication functional in at least oneorganism, convenient restriction endonuclease digestion sites andselectable markers appropriate for particular host cells. The vectorscan be transferred by a variety of means known to those of skill in theart into suitable host cells which then produce the desired DNA, RNA orpolypeptides.

Occasionally, sequencing or random reverse transcription errors willmask the presence of the appropriate open reading frame or regulatoryelement. In such cases, it is possible to determine the correct readingframe by attempting to express the polypeptide and determining the aminoacid sequence by standard peptide mapping and sequencing techniques.See, F. M. Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y. (1989). Additionally, the actual readingframe of a given nucleotide sequence may be determined by transfectionof host cells with vectors containing all three potential readingframes. Only those cells with the nucleotide sequence in the correctreading frame will produce a peptide of the predicted length.

The nucleotide sequences provided herein have been prepared by current,state-of-the-art, automated methods and as such may contain unidentifiednucleotides. These will not present a problem to those skilled in theart who wish to practice the invention. Several methods employingstandard recombinant techniques, described in J. Sambrook (supra) orperiodic updates thereof, may be used to complete the missing sequenceinformation. The same techniques used for obtaining a full lengthsequence, as described herein, may be used to obtain nucleotidesequences.

Expression of a particular cDNA may be accomplished by subcloning thecDNA into an appropriate expression vector and transfecting this vectorinto an appropriate expression host. The cloning vector used for thegeneration of the prostate tissue cDNA library can be used fortranscribing mRNA of a particular cDNA and contains a promoter forbeta-galactosidase, an amino-terminal met and the subsequent seven aminoacid residues of beta-galactosidase. Immediately following these eightresidues is an engineered bacteriophage promoter useful for artificialpriming and transcription, as well as a number of unique restrictionsites, including EcoRI, for cloning. The vector can be transfected intoan appropriate host strain of E. coli.

Induction of the isolated bacterial strain with isopropylthiogalactoside(IPTG) using standard methods will produce a fusion protein whichcontains the first seven residues of beta-galactosidase, about 15residues of linker and the peptide encoded within the cDNA. Since cDNAclone inserts are generated by an essentially random process, there isone chance in three that the included cDNA will lie in the correct framefor proper translation. If the cDNA is not in the proper reading frame,the correct frame can be obtained by deletion or insertion of anappropriate number of bases by well known methods including in vitromutagenesis, digestion with exonuclease III or mung bean nuclease, oroligonucleotide linker inclusion.

The cDNA can be shuttled into other vectors known to be useful forexpression of protein in specific hosts. Oligonucleotide primerscontaining cloning sites and segments of DNA sufficient to hybridize tostretches at both ends of the target cDNA, can be synthesized chemicallyby standard methods. These primers can then be used to amplify thedesired gene segments by PCR. The resulting new gene segments can bedigested with appropriate restriction enzymes under standard conditionsand isolated by gel electrophoresis. Alternately, similar gene segmentscan be produced by digestion of the cDNA with appropriate restrictionenzymes and filling in the missing gene segments with chemicallysynthesized oligonucleotides. Segments of the coding sequence from morethan one gene can be ligated together and cloned in appropriate vectorsto optimize expression of recombinant sequence.

Suitable expression hosts for such chimeric molecules include but arenot limited to, mammalian cells such as Chinese Hamster Ovary (CHO) andhuman embryonic kidney (HEK) 293 cells, insect cells such as Sf9 cells,yeast cells such as Saccharomyces cerevisiae and bacteria such as E.coli. For each of these cell systems, a useful expression vector mayalso include an origin of replication to allow propagation in bacteriaand a selectable marker such as the beta-lactamase antibiotic resistancegene to allow selection in bacteria. In addition, the vectors mayinclude a second selectable marker, such as the neomycinphosphotransferase gene, to allow selection in transfected eukaryotichost cells. Vectors for use in eukaryotic expression hosts may requirethe addition of 3′ poly A tail if the sequence of interest lacks poly A.

Additionally, the vector may contain promoters or enhancers whichincrease gene expression. Such promoters are host specific and include,but are not limited to, MMTV, SV40, or metallothionine promoters for CHOcells; trp, lac, tac or T7 promoters for bacterial hosts; or alphafactor, alcohol oxidase or PGH promoters for yeast. Adenoviral vectorswith or without transcription enhancers, such as the rous sarcoma virus(RSV) enhancer, may be used to drive protein expression in mammaliancell lines. Once homogeneous cultures of recombinant cells are obtained,large quantities of recombinantly produced protein can be recovered fromthe conditioned medium and analyzed using chromatographic methods wellknown in the art. An alternative method for the production of largeamounts of secreted protein involves the transfection of mammalianembryos and the recovery of the recombinant protein from milk producedby transgenic cows, goats, sheep, etc. Polypeptides and closely relatedmolecules may be expressed recombinantly in such a way as to facilitateprotein purification. One approach involves expression of a chimericprotein which includes one or more additional polypeptide domains notnaturally present on human polypeptides. Such purification-facilitatingdomains include, but are not limited to, metal-chelating peptides suchas histidine-tryptophan domains that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle, Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase fromInvitrogen (San Diego, Calif.) between the polypeptide sequence and thepurification domain may be useful for recovering the polypeptide.

Immunoassays

PS190 polypeptides, including fragments, derivatives, and analogsthereof, or cells expressing such polypeptides, can be utilized in avariety of assays, many of which are described herein, for the detectionof antibodies to prostate tissue. They also can be used as immunogens toproduce antibodies. These antibodies can be, for example, polyclonal ormonoclonal antibodies, chimeric, single chain and humanized antibodies,as well as Fab fragments, or the product of an Fab expression library.Various procedures known in the art may be used for the production ofsuch antibodies and fragments.

For example, antibodies generated against a polypeptide comprising asequence of the present invention can be obtained by direct injection ofthe polypeptide into an animal or by administering the polypeptide to ananimal such as a mouse, rabbit, goat or human. A mouse, rabbit or goatis preferred. The polypeptide is selected from the group consisting ofSEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO12, SEQUENCE ID NO 13, SEQUENCE ID NO 14, and fragments thereof. Theantibody so obtained then will bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies that bind the native polypeptide. Suchantibodies then can be used to isolate the polypeptide from test samplessuch as tissue suspected of containing that polypeptide. For preparationof monoclonal antibodies, any technique which provides antibodiesproduced by continuous cell line cultures can be used. Examples includethe hybridoma technique as described by Kohler and Milstein, Nature256:495-497 (1975), the trioma technique, the human B-cell hybridomatechnique as described by Kozbor et al., Immun. Today 4:72 (1983) andthe EBV-hybridoma technique to produce human monoclonal antibodies asdescribed by Cole, et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc, New York, N.Y., pp. 77-96 (1985). Techniquesdescribed for the production of single chain antibodies can be adaptedto produce single chain antibodies to immunogenic polypeptide productsof this invention. See, for example, U.S. Pat. No. 4,946,778, which isincorporated herein by reference.

Various assay formats may utilize the antibodies of the presentinvention, including “sandwich” immunoassays and probe assays. Forexample, the antibodies of the present invention, or fragments thereof,can be employed in various assay systems to determine the presence, ifany, of PS190 antigen in a test sample. For example, in a first assayformat, a polyclonal or monoclonal antibody or fragment thereof, or acombination of these antibodies, which has been coated on a solid phase,is contacted with a test sample, to form a first mixture. This firstmixture is incubated for a time and under conditions sufficient to formantigen/antibody complexes. Then, an indicator reagent comprising amonoclonal or a polyclonal antibody or a fragment thereof, or acombination of these antibodies, to which a signal generating compoundhas been attached, is contacted with the antigen/antibody complexes toform a second mixture. This second mixture then is incubated for a timeand under conditions sufficient to form antibody/antigen/antibodycomplexes. The presence of PS190 antigen in the test sample and capturedon the solid phase, if any, is determined by detecting the measurablesignal generated by the signal generating compound. The amount of PS190antigen present in the test sample is proportional to the signalgenerated.

In an alternative assay format, a mixture is formed by contacting: (1) apolyclonal antibody, monoclonal antibody, or fragment thereof, whichspecifically binds to PS190 antigen, or a combination of such antibodiesbound to a solid support; (2) the test sample; and (3) an indicatorreagent comprising a monoclonal antibody, polyclonal antibody, orfragment thereof, which specifically binds to a different PS190 antigen(or a combination of these antibodies) to which a signal generatingcompound is attached. This mixture is incubated for a time and underconditions sufficient to form antibody/antigen/antibody complexes. Thepresence, if any, of PS190 antigen present in the test sample andcaptured on the solid phase is determined by detecting the measurablesignal generated by the signal generating compound. The amount of PS190antigen present in the test sample is proportional to the signalgenerated.

In another assay format, one or a combination of at least two monoclonalantibodies of the invention can be employed as a competitive probe forthe detection of antibodies to PS190 antigen. For example, PS190polypeptides such as the recombinant antigens disclosed herein, eitheralone or in combination, are coated on a solid phase. A test samplesuspected of containing antibody to PS190 antigen then is incubated withan indicator reagent comprising a signal generating compound and atleast one monoclonal antibody of the invention for a time and underconditions sufficient to form antigen/antibody complexes of either thetest sample and indicator reagent bound to the solid phase or theindicator reagent bound to the solid phase. The reduction in binding ofthe monoclonal antibody to the solid phase can be quantitativelymeasured.

In yet another detection method, each of the monoclonal or polyclonalantibodies of the present invention can be employed in the detection ofPS190 antigens in tissue sections, as well as in cells, byimmunohistochemical analysis. Cytochemical analysis wherein theseantibodies are labeled directly (with, for example, fluorescein,colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) orare labeled by using secondary labeled anti-species antibodies (withvarious labels as exemplified herein) to track the histopathology ofdisease also are within the scope of the present invention.

In addition, these monoclonal antibodies can be bound to matricessimilar to CNBr-activated Sepharose and used for the affinitypurification of specific PS190 polypeptides from cell cultures orbiological tissues such as to purify recombinant and native PS190proteins.

The monoclonal antibodies of the invention also can be used for thegeneration of chimeric antibodies for therapeutic use, or other similarapplications.

The monoclonal antibodies or fragments thereof can be providedindividually to detect PS190 antigens. Combinations of the monoclonalantibodies (and fragments thereof) provided herein also may be usedtogether as components in a mixture or “cocktail” of at least one PS190antibody of the invention, along with antibodies which specifically bindto other PS190 regions, each antibody having different bindingspecificities. Thus, this cocktail can include the monoclonal antibodiesof the invention which are directed to PS190 polypeptides disclosedherein and other monoclonal antibodies specific to other antigenicdeterminants of PS190 antigens or other related proteins.

The polyclonal antibody or fragment thereof which can be used in theassay formats should specifically bind to a PS190 polypeptide or otherPS190 polypeptides additionally used in the assay. The polyclonalantibody used preferably is of mammalian origin such as, human, goat,rabbit or sheep polyclonal antibody which binds PS190 polypeptide. Mostpreferably, the polyclonal antibody is of rabbit origin. The polyclonalantibodies used in the assays can be used either alone or as a cocktailof polyclonal antibodies. Since the cocktails used in the assay formatsare comprised of either monoclonal antibodies or polyclonal antibodieshaving different binding specificity to PS190 polypeptides, they areuseful for the detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining thepredisposition to, diseases and conditions of the prostate, such asprostate cancer.

It is contemplated and within the scope of the present invention thatPS190 antigen may be detectable in assays by use of a recombinantantigen as well as by use of a synthetic peptide or purified peptide,which peptide comprises an amino acid sequence of PS190. The amino acidsequence of such a polypeptide is selected from the group consisting ofSEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO12, SEQUENCE ID NO 13, SEQUENCE ID NO 14, and fragments thereof. It alsois within the scope of the present invention that different synthetic,recombinant or purified peptides, identifying different epitopes ofPS190, can be used in combination in an assay for the detecting,diagnosing, staging, monitoring, prognosticating, preventing ortreating, or determining the predisposition to diseases and conditionsof the prostate, such as prostate cancer. In this case, all of thesepeptides can be coated onto one solid phase; or each separate peptidemay be coated onto separate solid phases, such as microparticles, andthen combined to form a mixture of peptides which can be later used inassays. Furthermore, it is contemplated that multiple peptides whichdefine epitopes from different antigens may be used for the detection,diagnosis, staging, monitoring, prognosis, prevention or treatment of,or determining the predisposition to, diseases and conditions of theprostate, such as prostate cancer. Peptides coated on solid phases orlabeled with detectable labels are then allowed to compete with thosepresent in a patient sample (if any) for a limited amount of antibody. Areduction in binding of the synthetic, recombinant, or purified peptidesto the antibody (or antibodies) is an indication of the presence ofPS190 antigen in the patient sample. The presence of PS190 antigenindicates the presence of prostate tissue disease, especially prostatecancer, in the patient. Variations of assay formats are known to thoseof ordinary skill in the art and many are discussed herein below.

In another assay format, the presence of anti-PS190 antibody and/orPS190 antigen can be detected in a simultaneous assay, as follows. Atest sample is simultaneously contacted with a capture reagent of afirst analyte, wherein said capture reagent comprises a first bindingmember specific for a first analyte attached to a solid phase and acapture reagent for a second analyte, wherein said capture reagentcomprises a first binding member for a second analyte attached to asecond solid phase, to thereby form a mixture. This mixture is incubatedfor a time and under conditions sufficient to form capture reagent/firstanalyte and capture reagent/second analyte complexes. These so-formedcomplexes then are contacted with an indicator reagent comprising amember of a binding pair specific for the first analyte labeled with asignal generating compound and an indicator reagent comprising a memberof a binding pair specific for the second analyte labeled with a signalgenerating compound to form a second mixture. This second mixture isincubated for a time and under conditions sufficient to form capturereagent/first analyte/indicator reagent complexes and capturereagent/second analyte/indicator reagent complexes. The presence of oneor more analytes is determined by detecting a signal generated inconnection with the complexes formed on either or both solid phases asan indication of the presence of one or more analytes in the testsample. In this assay format, recombinant antigens derived from theexpression systems disclosed herein may be utilized, as well asmonoclonal antibodies produced from the proteins derived from theexpression systems as disclosed herein. For example, in this assaysystem, PS190 antigen can be the first analyte. Such assay systems aredescribed in greater detail in EP Publication No. 0473065.

In yet other assay formats, the polypeptides disclosed herein may beutilized to detect the presence of antibody against PS190 antigen intest samples. For example, a test sample is incubated with a solid phaseto which at least one polypeptide such as a recombinant protein orsynthetic peptide has been attached. The polypeptide is selected fromthe group consisting of SEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE IDNO 11, SEQUENCE ID NO 12, SEQUENCE ID NO 13, SEQUENCE ID NO 14, andfragments thereof. These are reacted for a time and under conditionssufficient to form antigen/antibody complexes. Following incubation, theantigen/antibody complex is detected. Indicator reagents may be used tofacilitate detection, depending upon the assay system chosen. In anotherassay format, a test sample is contacted with a solid phase to which arecombinant protein produced as described herein is attached, and alsois contacted with a monoclonal or polyclonal antibody specific for theprotein, which preferably has been labeled with an indicator reagent.After incubation for a time and under conditions sufficient forantibody/antigen complexes to form, the solid phase is separated fromthe free phase, and the label is detected in either the solid or freephase as an indication of the presence of antibody against PS190antigen. Other assay formats utilizing the recombinant antigensdisclosed herein are contemplated. These include contacting a testsample with a solid phase to which at least one antigen from a firstsource has been attached, incubating the solid phase and test sample fora time and under conditions sufficient to form antigen/antibodycomplexes, and then contacting the solid phase with a labeled antigen,which antigen is derived from a second source different from the firstsource. For example, a recombinant protein derived from a first sourcesuch as E. coli is used as a capture antigen on a solid phase, a testsample is added to the so-prepared solid phase, and following standardincubation and washing steps as deemed or required, a recombinantprotein derived from a different source (i.e., non-E. coli) is utilizedas a part of an indicator reagent which subsequently is detected.Likewise, combinations of a recombinant antigen on a solid phase andsynthetic peptide in the indicator phase also are possible. Any assayformat which utilizes an antigen specific for PS190 produced or derivedfrom a first source as the capture antigen and an antigen specific forPS190 from a different second source is contemplated. Thus, variouscombinations of recombinant antigens, as well as the use of syntheticpeptides, purified proteins and the like, are within the scope of thisinvention. Assays such as this and others are described in U.S. Pat. No.5,254,458, which enjoys common ownership and is incorporated herein byreference.

Other embodiments which utilize various other solid phases also arecontemplated and are within the scope of this invention. For example,ion capture procedures for immobilizing an immobilizable reactioncomplex with a negatively charged polymer (described in EP publication0326100 and EP publication No. 0406473), can be employed according tothe present invention to effect a fast solution-phase immunochemicalreaction. An immobilizable immune complex is separated from the rest ofthe reaction mixture by ionic interactions between the negativelycharged poly-anion/immune complex and the previously treated, positivelycharged porous matrix and detected by using various signal generatingsystems previously described, including those described inchemiluminescent signal measurements as described in EPO Publication No.0 273,115.

Also, the methods of the present invention can be adapted for use insystems which utilize microparticle technology including automated andsemi-automated systems wherein the solid phase comprises a microparticle(magnetic or non-magnetic). Such systems include those described in, forexample, published EPO applications Nos. EP 0 425 633 and EP 0 424 634,respectively.

The use of scanning probe microscopy (SPM) for immunoassays also is atechnology to which the monoclonal antibodies of the present inventionare easily adaptable. In scanning probe microscopy, particularly inatomic force microscopy, the capture phase, for example, at least one ofthe monoclonal antibodies of the invention, is adhered to a solid phaseand a scanning probe microscope is utilized to detect antigen/antibodycomplexes which may be present on the surface of the solid phase. Theuse of scanning tunneling microscopy eliminates the need for labelswhich normally must be utilized in many immunoassay systems to detectantigen/antibody complexes. The use of SPM to monitor specific bindingreactions can occur in many ways. In one embodiment, one member of aspecific binding partner (analyte specific substance which is themonoclonal antibody of the invention) is attached to a surface suitablefor scanning. The attachment of the analyte specific substance may be byadsorption to a test piece which comprises a solid phase of a plastic ormetal surface, following methods known to those of ordinary skill in theart. Or, covalent attachment of a specific binding partner (analytespecific substance) to a test piece which test piece comprises a solidphase of derivatized plastic, metal, silicon, or glass may be utilized.Covalent attachment methods are known to those skilled in the art andinclude a variety of means to irreversibly link specific bindingpartners to the test piece. If the test piece is silicon or glass, thesurface must be activated prior to attaching the specific bindingpartner. Also, polyelectrolyte interactions may be used to immobilize aspecific binding partner on a surface of a test piece by usingtechniques and chemistries. The preferred method of attachment is bycovalent means. Following attachment of a specific binding member, thesurface may be further treated with materials such as serum, proteins,or other blocking agents to minimize non-specific binding. The surfacealso may be scanned either at the site of manufacture or point of use toverify its suitability for assay purposes. The scanning process is notanticipated to alter the specific binding properties of the test piece.

While the present invention discloses the preference for the use ofsolid phases, it is contemplated that the reagents such as antibodies,proteins and peptides of the present invention can be utilized innon-solid phase assay systems. These assay systems are known to thoseskilled in the art, and are considered to be within the scope of thepresent invention.

It is contemplated that the reagent employed for the assay can beprovided in the form of a test kit with one or more containers such asvials or bottles, with each container containing a separate reagent suchas a probe, primer, monoclonal antibody or a cocktail of monoclonalantibodies, or a polypeptide (e.g. recombinantly, synthetically producedor purified) employed in the assay. The polypeptide is selected from thegroup consisting of SEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE ID NO11, SEQUENCE ID NO 12, SEQUENCE ID NO 13, SEQUENCE ID NO 14, andfragments thereof. Other components such as buffers, controls and thelike, known to those of ordinary skill in art, may be included in suchtest kits. It also is contemplated to provide test kits which have meansfor collecting test samples comprising accessible body fluids, e.g.,blood, urine, saliva and stool. Such tools useful for collection(“collection materials”) include lancets and absorbent paper or clothfor collecting and stabilizing blood; swabs for collecting andstabilizing saliva; cups for collecting and stabilizing urine or stoolsamples. Collection materials, papers, cloths, swabs, cups and the like,may optionally be treated to avoid denaturation or irreversibleadsorption of the sample. The collection materials also may be treatedwith or contain preservatives, stabilizers or antimicrobial agents tohelp maintain the integrity of the specimens. Test kits designed for thecollection, stabilization and preservation of test specimens obtained bysurgery or needle biopsy are also useful. It is contemplated that allkits may be configured in two components which can be providedseparately; one component for collection and transport of the specimenand the other component for the analysis of the specimen. The collectioncomponent, for example, can be provided to the open market user whilethe components for analysis can be provided to others such as laboratorypersonnel for determination of the presence, absence or amount ofanalyte. Further, kits for the collection, stabilization andpreservation of test specimens may be configured for use by untrainedpersonnel and may be available in the open market for use at home withsubsequent transportation to a laboratory for analysis of the testsample.

E. coli bacteria (clone 2229812) has been deposited at the American TypeCulture Collection (A.T.C.C.), 12301 Parklawn Drive, Rockville, Md.20852, as of Feb. 29, 1997, under the terms of the Budapest Treaty andwill be maintained for a period of thirty (30) years from the date ofdeposit, or for five (5) years after the last request for the deposit,or for the enforceable period of the U.S. patent, whichever is longer.The deposit and any other deposited material described herein areprovided for convenience only, and are not required to practice thepresent invention in view of the teachings provided herein. The cDNAsequence in all of the deposited material is incorporated herein byreference. Clone 1548374 has been accorded A.T.C.C. Deposit No. 98591.

The present invention will now be described by way of examples, whichare meant to illustrate, but not to limit, the scope of the presentinvention.

EXAMPLES Example 1 Identification of Prostate Tissue Library PS190Gene-Specific Clones

A. Library Comparison of Expressed Sequence Tags (ESTs) or TranscriptImages

Partial sequences of cDNA clone inserts, so-called “expressed sequencetags” (ESTs), were derived from cDNA libraries made from prostate tumortissues, prostate non-tumor tissues and numerous other tissues, bothtumor and non-tumor and entered into a database (LIFESEQ™ database,available from Incyte Pharmaceuticals, Palo Alto, Calif.) as genetranscript images. See International Publication No. WO 95/20681. (Atranscript image is a listing of the number of EST's for each of therepresented genes in a given tissue library. ESTs sharing regions ofmutual sequence overlap are classified into clusters. A cluster isassigned a clone number from a representative 5′ EST. Often, a clusterof interest can be extended by comparing its consensus sequence withsequences of other EST's which did not meet the criteria for automatedclustering. The alignment of all available clusters and single ESTsrepresent a contig from which a consensus sequence is derived.) Thetranscript images then were evaluated to identify EST sequences thatwere representative primarily of the prostate tissue libraries. Thesetarget clones then were ranked according to their abundance (occurrence)in the target libraries and their absence from background libraries.Higher abundance clones with low background occurrence were given higherstudy priority. ESTs corresponding to the consensus sequence of PS190were found in 25% (6 of 24) of prostate tissue libraries. ESTscorresponding to the consensus sequence SEQUENCE ID NO 4 (or fragmentsthereof) were not found in any (0 of 399) of the other, non-prostate,libraries of the data base. Therefore, the consensus sequence orfragment thereof was found significantly more often in prostate thannon-prostate tissues. SEQUENCE ID NO 1, SEQUENCE ID NO 2, and SEQUENCEID NO 3, corresponding to overlapping clones 1548374, 2189842, and2273243, respectively, were identified for further study. Theserepresented the minimum number of clones that were needed to form thecontig and from which the consensus sequence provided herein (SEQUENCEID NO 4) was derived.

Sequences 17-21 contain additional sequence information to Sequences 1-8already provided.

B. Generation of a Consensus Sequence

The nucleotide sequences of clones 1548374 (SEQUENCE ID NO 1), 2189842(SEQUENCE ID NO 2), 2273243 (SEQUENCE ID NO 3 were entered in theSequencher™ Program (available from Gene Codes Corporation, Ann Arbor,Mich., in order to generate a nucleotide alignment (contig map) and thengenerate their consensus sequence (SEQUENCE ID NO 4). FIG. 1 shows thenucleotide sequence alignment of these clones and their resultantnucleotide consensus sequence (SEQUENCE ID NO 4). FIG. 2 presents thecontig map depicting the clones SEQUENCE ID NO 1, SEQUENCE ID NO 2, andSEQUENCE ID NO 3 forming overlapping regions of the PS190 gene and theresultant consensus nucleotide sequence (SEQUENCE ID NO 4) of theseclones in a graphic display. Following this, a three-frame translationwas performed on the consensus sequence (SEQUENCE ID NO 4). The secondforward frame was found to have an open reading frame encoding a 73residue amino acid sequence, which is presented as SEQUENCE ID NO 9.

Example 2 Sequencing of PS190 EST-Specific Clones

DNA sequences for clones which comprise the most upstream and downstreamESTs of the PS190 gene contig are determined using dideoxy terminationsequencing with either dye-labeled primers, dye terminators, orradiolabeled nucleotides, following known methods. (F. Sanger et al.,Proc. Natl. Acad. Sci. U.S.A. 74:5463 (1977)).

Because vectors such as pSPORT1 (Life Technologies, Gaithersburg, Md.)and pINCY (available from Incyte Pharmaceuticals, Inc., Palo Alto,Calif.) contain universal priming sites just adjacent to the 3′ and 5′ligation junctions of the inserts, the inserts are sequenced in bothdirections using universal primers, SEQUENCE ID NO 7 and SEQUENCE ID NO8 (New England Biolabs, Beverly, Mass. and Applied Biosystems Inc,Foster City, Calif., respectively). The sequencing reactions are run ona polyacrylamide denaturing gel, and the sequences are determined by anApplied Biosystems 377 Sequencer (available from Applied Biosystems,Foster City, Calif.) or other sequencing apparatus.

Example 3 Nucleic Acid Preparation

A. RNA Extraction from Tissue

Total RNA is isolated from solid prostate tissues or cells and fromnon-prostate tissues. Various methods are utilized, including but notlimited to the lithium chloride/urea technique, known and described inthe art (Kato et al., J. Virol. 61:2182-2191, [1987]), Ultraspec™(Biotecx Laboratories, Inc., Houston Tex.), and TRIzol™ (LifeTechnologies, Inc., Gaithersburg, Md.).

For northern blot analysis, the tissue is placed in a sterile conicaltube on ice and 10-15 volumes of 3 M LiCl, 6 M urea, 5 mM EDTA, 0.1 Mβ-mercaptoethanol, 50 mM Tris-HCl (pH 7.5) are added. The tissue ishomogenized with a Polytron® homogenizer (Brinkman Instruments, Inc.,Westbury, N.Y.) for 30-50 sec on ice. The solution is transferred to a15 ml plastic centrifuge tube and placed overnight at −20° C. The tubeis centrifuged for 90 min at 9,000×g at 0-4° C., and the supernatant isimmediately decanted. Then, 10 ml of 3 M LiCl are added, the tube isvortexed for 5 sec and centrifuged for 45 min at 11,000×g at 0-4° C.Decanting, resuspension in LiCl, and centrifugation are repeated. Thefinal pellet is air dried and resuspended in 2 ml of 1 mM EDTA, 0.5%SDS, 10 mM Tris (pH 7.5). Then, 20 μl of Proteinase K (20 mg/ml) areadded, and the solution is incubated for 30 min at 37° C. withoccasional mixing. One-tenth volume (0.22-0.25 ml) of 3 M NaCl is added,and the solution is vortexed before transfer into another tube whichcontains 2 ml of phenol/chloroform/isoamyl alcohol (PCI). The tube isvortexed for 1-3 sec and centrifuged for 20 min at 3,000×g at 10 °C. ThePCI extraction is repeated twice more, followed by two similarextractions with chloroform/isoamyl alcohol. The final aqueous solutionis transferred to a pre-chilled 15 ml corex glass tube containing 6 mlof 100% absolute ethanol, the tube is covered with parafilm and placedat −20° C. overnight. The tube is centrifuged for 30 min at 10,000×g at0-4° C., and the ethanol supernatant is decanted immediately. The RNApellet is washed four times with 10 ml of 75% ice-cold ethanol, followedeach time by centrifugation at 10,000×g for 10 min. The final pellet isair dried for 15 min at room temperature. The RNA is suspended in 0.5 mlof 10 mM Tris (pH 7.6), 1 mM EDTA, and its concentration is determinedspectrophotometrically. RNA samples are aliquoted and stored at −70° C.as ethanol precipitates.

The quality of the RNA is determined by agarose gel electrophoresis (seeExample 5) and staining with 0.5 μg/ml ethidium bromide for one hour.RNA samples that do not contain intact 28S/18S rRNAs are excluded fromthe study.

Alternatively, for RT-PCR analysis, 1 ml of Ultraspec RNA reagent isadded to 120 mg of pulverized tissue in a 2.0 ml polypropylene microfugetube, homogenized with a Polytron® homogenizer (Brinkman Instruments,Inc., Westbury, N.Y.) for 50 sec and left on ice for 5 min. Then, 0.2 mlof chloroform is added to each sample, followed by vortexing for 15 sec.The sample is left in ice for another 5 min, followed by centrifugationat 12,000×g for 15 min at 4° C. The upper layer is collected andtransferred to another RNase-free 2.0 ml microfuge tube. An equal volumeof isopropanol is added to each sample, and the solution is placed onice for 10 min. The sample is centrifuged at 12,000×g for 10 min at 4°C., and the supernatant is discarded. The remaining pellet is washedtwice with cold 75% ethanol, resuspended by vortexing, and theresuspended material is then re-pelleted by centrifugation at 7500×g for5 min at 4° C. Finally, the RNA pellet is dried in a speedvac for atleast 5 min and reconstituted in RNase-free water.

B. RNA Extraction from Blood Mononuclear Cells

Mononuclear cells are isolated from blood samples from patients bycentrifugation using Ficoll-Hypaque as follows. A 10 ml volume of wholeblood is mixed with an equal volume of RPMI Medium (Life Technologies,Gaithersburg, Md.). This mixture is then underlayed with 10 ml ofFicoll-Hypaque (Pharmacia, Piscataway, N.J.) and centrifuged for 30minutes at 200×g. The buffy coat containing the mononuclear cells isremoved, diluted to 50 ml with Dulbecco's PBS (Life Technologies,Gaithersburg, Md.) and the mixture centrifuged for 10 minutes at 200×g.After two washes, the resulting pellet is resuspended in Dulbecco's PBSto a final volume of 1 ml.

RNA is prepared from the isolated mononuclear cells as described by N.Kato et al., J. Virology 61: 2182-2191 (1987). Briefly, the pelletedmononuclear cells are brought to a final of 1 ml volume and then areresuspended in 250 μL of PBS and mixed with 2.5 ml of 3M LiCl, 6M urea,5 mM EDTA, 0.1M 2-mercaptoethanol, 50 mM Tris-HCl (pH 7.5). Theresulting mixture is homogenized and incubated at −20° C. overnight. Thehomogenate is spun at 8,000 RPM in a Beckman J2-21M rotor for 90 minutesat 0-4° C. The pellet is resuspended in 10 ml 3M LiCl by vortexing andthen spun at 10,000 RPM in a Beckman J2-21M rotor centrifuge for 45minutes at 0-4° C. The resuspending and pelleting steps then arerepeated. The pellet is resuspended in 2 ml of 1 mM EDTA, 0.5% SDS, 10mM Tris (pH 7.5) and 400 μg Proteinase K with vortexing and then it isincubated at 37° C. for 30 minutes with shaking. One tenth volume of 3MNaCl then is added and the vortexed mixture. Proteins are removed by twocycles of extraction with phenol/chloroform/isoamyl alcohol followed byone extraction with chloroform/isoamyl alcohol. RNA is precipitated bythe addition of 6 ml of ethanol followed by overnight incubation at −20°C. After the precipitated RNA is collected by centrifugation, the pelletis washed 4 times in 75% ethanol. The pelleted RNA is then dissolved in1 mM EDTA, 10 mM Tris-HCl (pH 7.5).

Non-prostate tissues are used as negative controls. The mRNA can befurther purified from total RNA by using commercially available kitssuch as oligo dT cellulose spin columns (RediCol™ from Pharmacia,Uppsala, Sweden) for the isolation of poly-adenylated RNA. Total or mRNAcan be dissolved in lysis buffer (5M guanidine thiocyanate, 0.1M EDTA,pH 7.0) for analysis in the ribonuclease protection assay.

C. RNA Extraction from polysomes

Tissue is minced in saline at 4° C. and mixed with 2.5 volumes of 0.8 Msucrose in a TK₁₅₀M (150 mM KCl, 5 mM MgCl₂, 50 mM Tris-HCl, pH 7.4)solution containing 6 mM 2-mercaptoethanol. The tissue is homogenized ina Teflon-glass Potter homogenizer with five strokes at 100-200 rpmfollowed by six strokes in a Dounce homogenizer, as described by B.Mechler, Methods in Enzymology 152:241-248 (1987). The homogenate thenis centrifuged at 12,000×g for 15 min at 4° C. to sediment the nuclei.The polysomes are isolated by mixing 2 ml of the supernatant with 6 mlof 2.5 M sucrose in TK₁₅₀M and layering this mixture over 4 ml of 2.5 Msucrose in TK₁₅₀M in a 38 ml polyallomer tube. Two additional sucroseTK₁₅₀M solutions are successively layered onto the extract fraction; afirst layer of 13 ml 2.05 M sucrose followed by a second layer of 6 mlof 1.3 M sucrose. The polysomes are isolated by centrifuging thegradient at 90,000×g for 5 h at 4° C. The fraction then is taken fromthe 1.3 M sucrose/2.05 M sucrose interface with a siliconized pasteurpipette and diluted in an equal volume of TE (10 mM Tris-HCl, pH 7.4, 1mM EDTA). An equal volume of 90° C. SDS buffer (1% SDS, 200 mM NaCl, 20mM Tris-HCl, pH 7.4) is added and the solution is incubated in a boilingwater bath for 2 min. Proteins next are digested with a Proteinase-Kdigestion (50 mg/ml) for 15 min at 37° C. The mRNA is purified with 3equal volumes of phenol-chloroform extractions followed by precipitationwith 0.1 volume of 2 M sodium acetate (pH 5.2) and 2 volumes of 100%ethanol at −20° C. overnight. The precipitated RNA is recovered bycentrifugation at 12,000×g for 10 min at 4° C. The RNA is dried andresuspended in TE (pH 7.4) or distilled water. The resuspended RNA thencan be used in a slot blot or dot blot hybridization assay to check forthe presence of PS190 mRNA (see Example 6).

The quality of nucleic acid and proteins is dependent on the method ofpreparation used. Each sample may require a different preparationtechnique to maximize isolation efficiency of the target molecule. Thesepreparation techniques are within the skill of the ordinary artisan.

Example 4 Ribonuclease Protection Assay

A. Synthesis of Labeled Complementary RNA (cRNA) Hybridization Probe andUnlabeled Sense Strand

Labeled antisense and unlabeled sense riboprobes are transcribed fromthe PS190 gene cDNA sequence which contains a 5′ RNA polymerase promotersuch as SP6 or T7. The sequence may be from a vector containing theappropriate PS190 cDNA insert, or from a PCR-generated product of theinsert using PCR primers which incorporate a 5′ RNA polymerase promotersequence. For example, the described plasmid, clone 1548374, containingthe PS190 gene cDNA sequence, flanked by opposed SP6 and T7 polymerasepromoters, is purified using Qiagen Plasmid Purification Kit (Qiagen,Chatsworth, Calif.). Then 10 μg of the plasmid are linearized by cuttingwith 10 U Dde I restriction enzyme for 1 h at 37° C. The linearizedplasmid is purified using QIAprep kits (Qiagen, Chatsworth, Calif.) andused for the synthesis of antisense transcript from the appropriate SP6or T7 promoter using the Riboprobe® in vitro Transcription System(Promega Corporation, Madison, Wis.), as described by the supplier'sinstructions, incorporating either 6.3 μM (alpha³²P) UTP (Amersham LifeSciences, Inc. Arlington Heights, Ill.) or 100-500 μM biotinylated UTPas a label. To generate the sense strand, 10 μg of the purified plasmidare cut with restriction enzymes 10 U XbaI and 10 U NotI, andtranscribed as above from the appropriate SP6 or T7 promoter. Both senseand antisense strands are isolated by spin column chromatography.Unlabeled sense strand is quantitated by UV absorption at 260 nm.

B. Hybridization of Labeled Probe to Target

Frozen tissue is pulverized to powder under liquid nitrogen and 100-500mg are dissolved in 1 ml of lysis buffer, available as a component ofthe Direct Protect™ Lysate RNase Protection kit (Ambion, Inc., Austin,Tex.). Further dissolution can be achieved using a tissue homogenizer.In addition, a dilution series of a known amount of sense strand inmouse liver lysate is made for use as a positive control. Finally, 45 μlof solubilized tissue or diluted sense strand is mixed directly witheither 1) 1×10⁵ cpm of radioactively labeled probe or 2) 250 pg ofnon-isotopically labeled probe in 5 μl of lysis buffer. Hybridization isallowed to proceed overnight at 37° C. See, T. Kaabache et al., Anal.Biochem. 232:225-230 (1995).

C. RNase Digestion

RNA that is not hybridized to probe is removed from the reaction as perthe Direct Protect™ protocol using a solution of RNase A and RNase T1for 30 min at 37° C., followed by removal of RNase by Proteinase-Kdigestion in the presence of sodium sarcosyl. Hybridized fragmentsprotected from digestion are then precipitated by the addition of anequal volume of isopropanol and placed at “70° C. for 3 h. Theprecipitates are collected by centrifugation at 12,000×g for 20 min.

D. Fragment Analysis

The precipitates are dissolved in denaturing gel loading dye (80%formamide, 10 mM EDTA (pH 8.0), 1 mg/ml xylene cyanol, 1 mg/mlbromophenol blue), heat denatured, and electrophoresed in 6%polyacrylamide TBE, 8 M urea denaturing gels. The gels are imaged andanalyzed using the STORM™ storage phosphor autoradiography system(Molecular Dynamics, Sunnyvale, Calif.). Quantitation of protectedfragment bands, expressed in femtograms (fg), is achieved by comparingthe peak areas obtained from the test samples to those from the knowndilutions of the positive control sense strand (see Section B, supra).The results are expressed in molecules of PS190 RNA/cell and as a imagerating score. In cases where non-isotopic labels are used, hybrids aretransferred from the gels to membranes (nylon or nitrocellulose) byblotting and then analyzed using detection systems that employstreptavidin alkaline phosphatase conjugates and chemiluminesence orchemifluoresence reagents. High level expression of mRNA correspondingto a sequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof, indicate the presence of PS190 mRNA(s), suggestinga diagnosis of a prostate tissue disease or condition, such as prostatecancer.

Example 5 Northern Blotting

The northern blot technique is used to identify a specific size RNAfragment from a complex population of RNA using gel electrophoresis andnucleic acid hybridization. Northern blotting is well-known technique inthe art. Briefly, 5-10 μg of total RNA (see Example 3) are incubated in15 μl of a solution containing 40 mM morphilinopropanesulfonic acid(MOPS) (pH 7.0), 10 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde,50% v/v formamide for 15 min at 65° C. The denatured RNA is mixed with 2μl of loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromophenol blue,0.4% xylene cyanol) and loaded into a denaturing 1.0% agarose gelcontaining 40 mM MOPS (pH 7.0), 10 mM sodium acetate, 1 mM EDTA and 2.2M formaldehyde. The gel is electrophoresed at 60 V for 1.5 h and rinsedin RNAse free water. RNA is transferred from the gel onto nylonmembranes (Brightstar-Plus, Ambion, Inc., Austin, Tex.) for 1.5 hoursusing the downward alkaline capillary transfer method (Chomczynski,Anal. Biochem. 201:134-139, 1992). The filter is rinsed with 1×SSC, andRNA is crosslinked to the filter using a Stratalinker (Stratagene, Inc.,La Jolla, Calif.) on the autocrosslinking mode and dried for 15 min. Themembrane is then placed into a hybridization tube containing 20 ml ofpreheated prehybridization solution (5×SSC, 50% formamide, 5×Denhardt'ssolution, 100 μg/ml denatured salmon sperm DNA) and incubated in a 42°C. hybridization oven for at least 3 hr. While the blot isprehybridizing, a ³²P-labeled random-primed probe is generated using thePS190 insert fragment (obtained by digesting clone 1548374 with XbaI andNotI) using Random Primer DNA Labeling System (Life Technologies, Inc.,Gaithersburg, Md.) according to the manufacturer's instructions. Half ofthe probe is boiled for 10 min, quick chilled on ice and added to thehybridization tube. Hybridization is carried out at 42° C. for at least12 hr. The hybridization solution is discarded and the filter is washedin 30 ml of 3×SSC, 0.1% SDS at 42° C. for 15 min, followed by 30 ml of3×SSC, 0.1% SDS at 42° C. for 15 min. The filter is wrapped in saranwrap, exposed to Kodak XAR-Omat film for 8-96 hr, and the film isdeveloped for analysis. High level of expression of mRNA correspondingto a sequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof, is an indication of the presence of PS190 mRNA,suggesting a diagnosis of a prostate tissue disease or condition, suchas prostate cancer.

Example 6 Dot Blot/Slot Blot

Dot and slot blot assays are quick methods to evaluate the presence of aspecific nucleic acid sequence in a complex mix of nucleic acid. Toperform such assays, up to 50 μg of RNA is mixed in 50 μl of 50%formamide, 7% formaldehyde, 1×SSC, incubated 15 min at 68° C., and thencooled on ice. Then, 100 μl of 20×SSC is added to the RNA mixture andloaded under vacuum onto a manifold apparatus that has a preparednitrocellulose or nylon membrane. The membrane is soaked in water,20×SSC for 1 hour, placed on two sheets of 20×SSC prewet Whatman #3filter paper, and loaded into a slot blot or dot blot vacuum manifoldapparatus. The slot blot is analyzed with probes prepared and labeled asdescribed in Example 4, supra. Detection of mRNA corresponding to asequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof, is an indication of the presence of PS190,suggesting a diagnosis of a prostate tissue disease or condition, suchas prostate cancer.

Other methods and buffers which can be utilized in the methods describedin Examples 5 and 6, but not specifically detailed herein, are known inthe art and are described in J. Sambrook et al., supra which isincorporated herein by reference.

Example 7 In Situ Hybridization

This method is useful to directly detect specific target nucleic acidsequences in cells using detectable nucleic acid hybridization probes.

Tissues are prepared with cross-linking fixative agents such asparaformaldehyde or glutaraldehyde for maximum cellular RNA retention.See, L. Angerer et al., Methods in Cell Biol. 35:37-71 (1991). Briefly,the tissue is placed in greater than 5 volumes of 1% glutaraldehyde in50 mM sodium phosphate, pH 7.5 at 4° C. for 30 min. The solution ischanged with fresh glutaraldehyde solution (1% glutaraldehyde in 50 mMsodium phosphate, pH 7.5) for a further 30 min fixing. The fixingsolution should have an osmolality of approximately 0.375% NaCl. Thetissue is washed once in isotonic NaCl to remove the phosphate.

The fixed tissues then are embedded in paraffin as follows. The tissueis dehydrated though a series of ethanol concentrations for 15 min each:50% (twice), 70% (twice), 85%, 90% and then 100% (twice). Next, thetissue is soaked in two changes of xylene for 20 min each at roomtemperature. The tissue is then soaked in two changes of a 1:1 mixtureof xylene and paraffin for 20 min each at 60° C.; and then in threefinal changes of paraffin for 15 min each.

Next, the tissue is cut in 5 μm sections using a standard microtome andplaced on a slide previously treated with a tissue adhesive such as3-aminopropyltriethoxysilane.

Paraffin is removed from the tissue by two 10 min xylene soaks andrehydrated in a series of ethanol concentrations: 99% twice, 95%, 85%,70%, 50%, 30%, and then distilled water twice. The sections arepre-treated with 0.2 M HCl for 10 min and permeabilized with 2 μg/mlProteinase-K at 37° C. for 15 min.

Labeled riboprobes transcribed from the PS190 gene plasmid (see Example4) are hybridized to the prepared tissue sections and incubatedovernight at 56° C. in 3×standard saline extract and 50% formamide.Excess probe is removed by washing in 2×standard saline citrate and 50%formamide followed by digestion with 100 μg/ml RNase A at 37° C. for 30min. Fluorescence probe is visualized by illumination with ultraviolet(UV) light under a microscope. Fluorescence in the cytoplasm isindicative of PS190 mRNA. Alternatively, the sections can be visualizedby autoradiography.

Example 8 Reverse Transcription PCR

A. One Step RT-PCR Assay

Target-specific primers are designed to detect the above-describedtarget sequences by reverse transcription PCR using methods known in theart. One step RT-PCR is a sequential procedure that performs both RT andPCR in a single reaction mixture. The procedure is performed in a 200 μlreaction mixture containing 50 mM (N,N,-bis[2-Hydroxyethyl]glycine), pH8.15, 81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1mM ethylene diaminetetraacetic acid, 0.02 mg/ml NaN₃, 8% w/v glycerol,150 μM each of dNTP, 0.25 μM each primer, 5U rTth polymerase, 3.25 mMMn(OAc)₂ and 5 μl of target RNA (see Example 3). Since RNA and the rTthpolymerase enzyme are unstable in the presence of Mn(OAc)₂, the Mn(OAc)₂should be added just before target addition. Optimal conditions for cDNAsynthesis and thermal cycling readily can be determined by those skilledin the art. The reaction is incubated in a Perkin-Elmer Thermal Cycler480. Optimal conditions for cDNA synthesis and thermal cycling canreadily be determined by those skilled in the art. Conditions which maybe found useful include cDNA synthesis at 60°-70° C. for 15-45 min and30-45 amplification cycles at 94° C., 1 min; 55°-70° C., 1 min; 72° C.,2 min. One step RT-PCR also may be performed by using a dual enzymeprocedure with Taq polymerase and a reverse transcriptase enzyme, suchas MMLV or AMV RT enzymes.

B. Traditional RT-PCR

Alternatively, a traditional two-step RT-PCR reaction may be performed,as described by K. Q. Hu et al., Virology 181:721-726 (1991), asfollows. The extracted mRNA is transcribed in a 25 μl reaction mixturecontaining 10 mM Tris-HCl, pH 8.3, 5 mM MgCl₂, 500 μM dNTP, 20 U RNasin,1 μM antisense primer and 25 U AMV (avian myeloblastosis virus) or MMLV(Moloney murine leukemia virus) reverse transcriptase. Reversetranscription is performed at 37-45° C. for 30-60 min, followed byfurther incubation at 95° C. for 5 min to inactivate the RT. PCR isperformed using 10 μl of the cDNA reaction in a final PCR reactionvolume of 50 μl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mMMgCl₂, 200 μM dNTP, 0.5 μM of each primer and 2.5 U of Taq polymerase.Optimal conditions for cDNA synthesis and thermal cycling can be readilydetermined by those skilled in the art. The reaction is incubated in aPerkin-Elmer Thermal Cycler 480 or other comparable instrument.Conditions which may be found useful include 30-45 cycles ofamplification (94° C., 1 min; 55-70° C., 1 min; 72° C., 2 min), finalextension (72° C., 10 min) and soak at 4° C.

C. PCR Fragment Analysis

The correct products then can be verified by size determination usinggel electrophoresis with fluorescent intercalators, such as SYBR® GreenI (Molecular Probes, Eugene, Oreg.) and imaged using a STORM imagingsystem, or also verified by Southern, dot or slot blot analysis using alabeled probe against the internal sequences of the PCR product. Theprobes also may be polynucleotides analogs, such as morpholinos orpeptide nucleic acids analogs (PNAs). Detection of a product comprisinga sequence selected from the group consisting of SEQUENCE ID NO 1,SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, and fragments orcomplements thereof, is indicative of the presence of PS190 mRNA(s),suggesting a diagnosis of a prostate tissue disease or condition, suchas prostate cancer.

Example 9 OH-PCR

A. Probe selection and Labeling

Target-specific primers and probes are designed to detect theabove-described target sequences by oligonucleotide hybridization PCR.International Publication Nos WO 92/10505, published Jun. 25, 1992, andWO 92/11388, published Jul. 9, 1992, teach methods for labelingoligonucleotides at their 5′ and 3′ ends, respectively. According to oneknown method for labeling an oligonucleotide, a label-phosphoramiditereagent is prepared and used to add the label to the oligonucleotideduring its synthesis. For example, see N. T. Thuong et al., Tet. Letters29(46):5905-5908 (1988); or J. S. Cohen et al., published U.S. patentapplication Ser. No. 07/246,688 (NTIS ORDER No. PAT-APPL-7-246,688)(1989). Preferably, probes are labeled at their 3′ end to preventparticipation in PCR and the formation of undesired extension products.For one step OH-PCR, the probe should have a T_(M) at least 15° C. belowthe T_(M) of the primers. The primers and probes are utilized asspecific binding members, with or without detectable labels, usingstandard phosphoramidite chemistry and/or post-synthetic labelingmethods which are well-known to one skilled in the art.

B. One Step Oligo Hybridization PCR

OH-PCR is performed on a 200 μl reaction containing 50 mM(N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 mM KOH,0.01 mg/ml bovine serum albumin, 0.1 mM ethylene diaminetetraaceticacid, 0.02 mg/ml NaN₃, 8% w/v glycerol, 150 μM each of dNTP, 0.25 μMeach primer, 3.75 nM probe, 5U rTth polymerase, 3.25 mM Mn(OAc)₂ and 5μl blood equivalents of target (see Example 3). Since RNA and the rTthpolymerase enzyme are unstable in the presence of Mn(OAc)₂, the Mn(OAc)₂should be added just before target addition. The reaction is incubatedin a Perkin-Elmer Thermal Cycler 480. Optimal conditions for cDNAsynthesis and thermal cycling can be readily determined by those skilledin the art. Conditions which may be found useful include cDNA synthesis(60° C., 30 min), 30-45 amplification cycles (94° C., 40 sec; 55-70° C.,60 sec), oligo-hybridization (97° C., 5 min; 15° C., 5 min; 15° C.soak). The correct reaction product contains at least one of the strandsof the PCR product and an internally hybridized probe.

C. OH-PCR Product Analysis

Amplified reaction products are detected on an LCx® analyzer system(available from Abbott Laboratories, Abbott Park, Ill.). Briefly, thecorrect reaction product is captured by an antibody labeledmicroparticle at a capturable site on either the PCR product strand orthe hybridization probe, and the complex is detected by binding of adetectable antibody conjugate to either a detectable site on the probeor the PCR strand. Only a complex containing a PCR strand hybridizedwith the internal probe is detectable. The detection of this complexthen is indicative of the presence of PS190 mRNA, suggesting a diagnosisof a prostate disease or condition, such as prostate cancer.

Many other detection formats exist which can be used and/or modified bythose skilled in the art to detect the presence of amplified ornon-amplified PS190-derived nucleic acid sequences including, but notlimited to, ligase chain reaction (LCR, Abbott Laboratories, AbbottPark, Ill.); Q-beta replicase (Gene-Trak™, Naperville, Ill.), branchedchain reaction (Chiron, Emeryville, Calif.) and strand displacementassays (Becton Dickinson, Research Triangle Park, N.C.).

Example 10 Synthetic Peptide Production

Synthetic peptides are modeled and then prepared based upon thepredicted amino acid sequence of the PS190 polypeptide consensussequence (see example 1). Peptides modeled for PS190 include SEQUENCE IDNO 10, SEQUENCE ID NO 11, SEQUENCE ID NO 12, SEQUENCE ID NO 13, SEQUENCEID NO 14, and fragments thereof derived from SEQUENCE ID NO 9. Allpeptides are synthesized on a Symphony Peptide Synthesizer (availablefrom Rainin Instrument Co., Emeryville Calif.) or similar instrument,using FMOC chemistry, standard cycles and in-situ HBTU activation.Cleavage and deprotection conditions are as follows: a volume of 2.5 mlof cleavage reagent (77.5% v/v trifluoroacetic acid, 15% v/vethanedithiol, 2.5% v/v water, 5% v/v thioanisole, 1-2% w/v phenol) isadded to the resin, and agitated at room temperature for 2-4 hours. Thenthe filtrate is removed and the peptide is precipitated from thecleavage reagent with cold diethyl ether. Each peptide is filtered,purified via reverse-phase preparative HPLC using awater/acetonitrile/0.1% TFA gradient, and lyophilized. The product isconfirmed by mass spectrometry (see Example 12).

Disulfide bond formation is accomplished using auto-oxidationconditions, as follows: the peptide is dissolved in a minimum amount ofDMSO (approximately 10 ml) before adding buffer (0.1 M Tris-HCl, pH 6.2)to a concentration of 0.3-0.8 mg/ml. The reaction is monitored by HPLCuntil complete formation of the disulfide bond, followed byreverse-phase preparative HPLC using a water/acetonitrile/0.1% TFAgradient and lyophilization. The product then is confirmed by massspectrometry (see Example 12).

The purified peptides can be conjugated to Keyhole Limpet Hemocyanin orother immunoreactive molecule with glutaraldehyde, mixed with adjuvant,and injected into animals.

Example 11a Expression of Protein in a Cell Line Using Plasmid 577

A. Construction of a PS190 Expression Plasmid

Plasmid 577, described in U.S. patent application Ser. No. 08/478,073,filed Jun. 7, 1995 and incorporated herein by reference, has beenconstructed for the expression of secreted antigens in a permanent cellline. This plasmid contains the following DNA segments: (a) a 2.3 Kbfragment of pBR322 containing bacterial beta-lactamase and origin of DNAreplication; (b) a 1.8 Kb cassette directing expression of a neomycinresistance gene under control of HSV- 1 thymidine kinase promoter andpoly-A addition signals; (c) a 1.9 Kb cassette directing expression of adihydrofolate reductase gene under the control of an SV-40 promoter andpoly-A addition signals; (d) a 3.5 Kb cassette directing expression of arabbit immunoglobulin heavy chain signal sequence fused to a modifiedhepatitis C virus (HCV) E2 protein under the control of the Simian Virus40 T-Ag promoter and transcription enhancer, the hepatitis B virussurface antigen (HBsAg) enhancer I followed by a fragment of HerpesSimplex Virus-1 (HSV-1) genome providing poly-A addition signals; and(e) a residual 0.7 Kb fragment of Simian Virus 40 genome late region ofno function in this plasmid. All of the segments of the vector wereassembled by standard methods known to those skilled in the art ofmolecular biology.

Plasmids for the expression of secretable PS190 proteins are constructedby replacing the hepatitis C virus E2 protein coding sequence in plasmid577 with that of a PS190 polynucleotide sequence selected from the groupconsisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3,SEQUENCE ID NO 4, and fragments or complements thereof, as follows.Digestion of plasmid 577 with XbaI releases the hepatitis C virus E2gene fragment. The resulting plasmid backbone allows insertion of thePS190 cDNA insert downstream of the rabbit immunoglobulin heavy chainsignal sequence which directs the expressed proteins into the secretorypathway of the cell. The PS190 cDNA fragment is generated by PCR usingstandard procedures. Encoded in the sense PCR primer sequence is an XbaIsite, immediately followed by a 12 nucleotide sequence that encodes theamino acid sequence Ser-Asn-Glu-Leu (“SNEL”) to promote signal proteaseprocessing, efficient secretion and final product stability in culturefluids. Immediately following this 12 nucleotide sequence the primercontains nucleotides complementary to template sequences encoding aminoacids of the PS190 gene. The antisense primer incorporates a sequenceencoding the following eight amino acids just before the stop codons:Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQUENCE ID NO 15). Within thissequence is incorporated a recognition site to aid in analysis andpurification of the PS190 protein product. A recognition site (termed“FLAG”) that is recognized by a commercially available monoclonalantibody designated anti-FLAG M2 (Eastman Kodak, Co., New Haven, Conn.)can be utilized, as well as other comparable sequences and theircorresponding antibodies. For example, PCR is performed using GeneAmp®reagents obtained from Perkin-Elmer-Cetus, as directed by the supplier'sinstructions. PCR primers are used at a final concentration of 0.5 μM.PCR is performed on the PS190 plasmid template in a 100 μl reaction for35 cycles (94° C., 30 seconds; 55° C., 30 seconds; 72° C., 90 seconds)followed by an extension cycle of 72° C. for 10 min.

B. Transfection of Dihydrofolate Reductase Deficient Chinese HamsterOvary Cells

The plasmid described supra is transfected into CHO/dhfr-cells (DXB-111,Uriacio et al., PNAS 77:4451-4466 (1980)). These cells are availablefrom the A.T.C.C., 12301 Parklawn Drive, Rockville, Md. 20852, underAccession No. CRL 9096. Transfection is carried out using the cationicliposome-mediated procedure described by P. L. Felgner et al., PNAS84:7413-7417 (1987). Particularly, CHO/dhfr-cells are cultured in Ham'sF-12 media supplemented with 10% fetal calf serum, L-glutamine (1 mM)and freshly seeded into a flask at a density of 5-8×10⁵ cells per flask.The cells are grown to a confluency of between 60 and 80% fortransfection. Twenty micrograms (20 μg) of plasmid DNA is added to 1.5ml of Opti-MEM I medium and 100 μl of Lipofectin Reagent (Gibco-BRL;Grand Island, N.Y.) are added to a second 1.5 ml portion of Opti-MEM Imedia. The two solutions are mixed and incubated at room temperature for20 min. After the culture medium is removed from the cells, the cellsare rinsed 3 times with 5 ml of Opti-MEM I medium. The Opti-MEMI-Lipofection-plasmid DNA solution then is overlaid onto the cells. Thecells are incubated for 3 h at 37° C., after which time the Opti-MEMI-Lipofectin-DNA solution is replaced with culture medium for anadditional 24 h prior to selection.

C. Selection and Amplification

One day after transfection, cells are passaged 1:3 and incubated withdhfr/G418 selection medium (hereafter, “F-12 minus medium G”). Selectionmedium is Ham's F-12 with L-glutamine and without hypoxanthine,thymidine and glycine (JRH Biosciences, Lenexa, Kans.) and 300 μg per mlG418 (Gibco-BRL; Grand Island, N.Y.). Media volume-to-surface arearatios of 5 ml per 25 cm² are maintained. After approximately two weeks,DHFR/G418 cells are expanded to allow passage and continuous maintenancein F-12 minus medium G.

Amplification of each of the transfected PS190 cDNA sequences isachieved by stepwise selection of DHFR⁺, G418⁺ cells with methotrexate(reviewed by R. Schimke, Cell 37:705-713 [1984]). Cells are incubatedwith F-12 minus medium G containing 150 nM methotrexate (MTX) (Sigma,St. Louis, Mo.) for approximately two weeks until resistant coloniesappear. Further gene amplification is achieved by selection of 150 nMadapted cells with 5 μM MTX.

D. Antigen Production

F-12 minus medium G supplemented with 5 μM MTX is overlaid onto justconfluent monolayers for 12 to 24 h at 37° C. in 5% CO₂. The growthmedium is removed and the cells are rinsed 3 times with Dulbecco'sphosphate buffered saline (PBS) (with calcium and magnesium) (Gibco-BRL;Grand Island, N.Y.) to remove the remaining media/serum which may bepresent. Cells then are incubated with VAS custom medium (VAS customformulation with L-glutamine with HEPES without phenol red, availablefrom JRH Bioscience; Lenexa, Kans., product number 52-08678P), for 1 hat 37° C. in 5% CO₂. Cells then are overlaid with VAS for production at5 ml per T flask. Medium is removed after seven days of incubation,retained, and then frozen to await purification with harvests 2, 3 and4. The monolayers are overlaid with VAS for 3 more seven day harvests.

E. Analysis of Prostate Tissue Gene PS190 Antigen Expression

Aliquots of VAS supernatants from the cells expressing the PS190 proteinconstruct are analyzed, either by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) using standard methods and reagents known in the art (Laemmlidiscontinuous gels), or by mass spectrometry.

F. Purification

Purification of the PS190 protein containing the FLAG sequence isperformed by immunoaffinity chromatography using an affinity matrixcomprising anti-FLAG M2 monoclonal antibody covalently attached toagarose by hydrazide linkage (Eastman Kodak Co., New Haven, Conn.).Prior to affinity purification, protein in pooled VAS medium harvestsfrom roller bottles is exchanged into 50 mM Tris-HCl (pH 7.5), 150 mMNaCl buffer using a Sephadex G-25 (Pharmacia Biotech Inc., Uppsala,Sweden) column. Protein in this buffer is applied to the anti-FLAG M2antibody affinity column. Non-binding protein is eluted by washing thecolumn with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl buffer. Bound proteinis eluted using an excess of FLAG peptide in 50 mM Tris-HCl (pH 7.5),150 mM NaCl. The excess FLAG peptide can be removed from the purifiedPS190 protein by gel electrophoresis or HPLC.

Although plasmid 577 is utilized in this example, it is known to thoseskilled in the art that other comparable expression systems, such asCMV, can be utilized herein with appropriate modifications in reagentand/or techniques and are within the skill of the ordinary artisan.

The largest cloned insert containing the coding region of the PS190 geneis then sub-cloned into either (i) a eukaryotic expression vector whichmay contain, for example, a cytomegalovirus (CMV) promoter and/orprotein fusible sequences which aid in protein expression and detection,or (ii) a bacterial expression vector containing a superoxide-dismutase(SOD) and CMP-KDO synthetase (CKS) or other protein fusion gene forexpression of the protein sequence. Methods and vectors which are usefulfor the production of polypeptides which contain fusion sequences of SODare described in EPO 0196056, published Oct. 1, 1986, which isincorporated herein by reference and those containing fusion sequencesof CKS are described in EPO Publication No. 0331961, published Sep. 13,1989, which publication is also incorporated herein by reference. Thisso-purified protein can be used in a variety of techniques, includingbut not limited to animal immunization studies, solid phaseimmunoassays, etc.

Example 11b Expression of Protein in a Cell Line Using pcDNA3.1/Myc-His

A. Construction of a PS190 Expression Plasmid

Plasmid pcDNA3.1/Myc-His (Cat.# V855-20, Invitrogen, Carlsbad, Calif.)has been constructed, in the past, for the expression of secretedantigens by most mammalian cell lines. Expressed protein inserts arefused to a myc-his peptide tag. The myc-his tag is a 21 residue aminoacid sequence having the following sequence:Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-Asn-Met-His-Thr-Glu-His-His-His-His-His-His (SEQUENCE ID NO 16) and comprises amyc epitope and a polyhistidine sequence which are useful for thepurification of an expressed fusion protein using either anti-myc oranti-his affinity columns, or metalloprotein binding columns.

Plasmids for the expression of secretable PS190 proteins are constructedby inserting a PS190 polynucleotide sequence selected from the groupconsisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, SEQUENCE ID NO 3,SEQUENCE ID NO 4, and fragments or complements thereof. Prior toconstruction of a PS190 expression plasmid, the PS190 cDNA sequence isfirst cloned into a pCR®-Blunt vector as follows.

The PS190 cDNA fragment is generated by PCR using standard procedures.For example, PCR is performed using Stratagene® reagents obtained fromStratagene, La Jolla, Calif., as directed by the supplier'sinstructions. PCR primers are used at a final concentration of 0.5 μM.PCR using 5 U of pfu polymerase (Stratagene) is performed on the PS190plasmid template (see Example 2) in a 50 μl reaction for 30 cycles (94°C., 1 min; 65° C., 1.5 min; 72° C., 3 min) followed by an extensioncycle at 72° C. for 8 min. The sense PCR primer sequence comprisesnucleotides which are either complementary to the pINCY vector directlyupstream of the PS190 gene insert or which incorporate a 5′ EcoRIrestriction site, an adjacent downstream protein translation consensusinitiator, and a 3′ nucleic acid sequence which is the same sense as the5′-most end of the PS190 cDNA insert. The antisense primer incorporatesa 5′ NotI restriction sequence and a sequence complementary to the 3′end of the PS190 cDNA insert just upstream of the 3′-most, in-frame stopcodon. Five microliters (5 μl) of the resulting blunt-ended PCR productare ligated into 25 ng of linearized pCR®-Blunt vector (Invitrogen,Carlsbad, Calif.) interrupting the lethal ccdB gene of the vector. Theresulting ligated vector is transfected into TOP10 E. coli (Invitrogen,Carlsbad, Calif.) using a One Shot™ transformation kit (Invitrogen,Carlsbad, Calif.) following the supplier's directions. The transfectedcells are grown on LB-Kan (50 μg/ml kanamycin) selection plates at 37°C. Only cells containing a plasmid with an interrupted ccdB gene willgrow after transfection (Grant, S. G. N., PNAS USA 87:4645-4649 (1990)).Transfected colonies are picked and grown up in 3 ml of LB-Kan broth at37° C. Plasmid DNA is isolated using a QIAprep® (Qiagen Inc., SantaClarita, Calif.) procedure, as directed by the supplier's instructions.The DNA is cut with EcoRI or SnaBI, and NotI restriction enzymes torelease the PS190 insert fragment. The fragment is run on 1% Seakem® LEagarose/0.5 μg/ml ethidium bromide/TE gel, visualized by UV irradiation,excised and purified using QIAquick™ (Qiagen Inc., Santa Clarita,Calif.) procedures, as directed by the supplier's instructions.

The pcDNA3.1/Myc-His plasmid DNA is linearized by digestion with EcoRIor SnaBI, and NotI in the polylinker region of the plasmid DNA. Theresulting plasmid DNA backbone allows insertion of the PS190 purifiedcDNA fragment, supra, downstream of a CMV promoter which directsexpression of the proteins in mammalian cells. The ligated plasmid istransfected into DH5 alpha™ cells (GibcoBRL, Gaithersburg, Md.) asdirected by the supplier's instructions. Briefly, 10 ng ofpcDNA3.1/Myc-His containing a PS190 insert is added to 50 μl ofcompetent DH5 alpha cells, and the contents are mixed gently. Themixture is incubated on ice for 30 min, heat shocked for 20 sec at 37°C., and placed on ice for an additional 2 min. Upon addition of 0.95 mlof LB medium, the mixture is incubated for 1 h at 37° C. while shakingat 225 rpm. The transfected cells are then plated onto 100 mm LB/Amp (50μg/ml ampicillin) plates and grown at 37° C. Colonies are picked andgrown in 3 ml of LB/Amp broth. Plasmid DNA is purified using a QIAprep®kit. Presence of the insert is confirmed using techniques known to thoseskilled in the art including, but not limited to, restriction digestionand gel analysis. See, e.g., J. Sambrook et al., supra.

B. Transfection of Human Embryonic Kidney 293 Cells

The PS190 expression plasmid described supra is transfected into HEK293cells (F. L. Graham et al., J. Gen. Vir. 36:59-72 (1977)). These cellsare available from the A.T.C.C., 12301 Parklawn Drive, Rockville, Md.20852, under Accession No. CRL 1573. Transfection is carried out usingthe cationic lipofectamine-mediated procedure described by P.Hawley-Nelson et al., Focus 15:73 (1993). Particularly, HEK293 cells arecultured in 10 ml DMEM media supplemented with 10% fetal bovine serum(FBS), L-glutamine (2 mM) and freshly seeded into 100 mm culture platesat a density of 9×10⁶ cells per plate. The cells are grown at 37° C. toa confluency of between 70% and 80% for transfection. Eight micrograms(8 μg) of plasmid DNA is added to 800 μl of Opti-MEM I® medium(Gibco-BRL, Grand Island, N.Y.), and 48-96 μl of Lipofectamine™ Reagent(Gibco-BRL, Grand Island, N.Y.) is added to a second 800 μl portion ofOpti-MEM I® media. The two solutions are mixed and incubated at roomtemperature for 15-30 min. After the culture medium is removed from thecells, the cells are washed once with 10 ml of serum-free DMEM. TheOpti-MEM I®-Lipofectamine-plasmid DNA solution is diluted in 6.4 ml ofserum-free DMEM and then overlaid onto the cells. The cells areincubated for 5 h at 37° C., after which time, an additional 8 ml ofDMEM with 20% FBS is added. After 18-24 h, the old medium is aspirated,and the cells are overlaid with 5 ml of fresh DMEM with 10% FBS.Supernatants and cell extracts are analyzed for PS190 gene activity 72 hafter transfection.

C. Analysis of Prostate Tissue Gene PS190 Antigen Expression

The culture supernatant, supra, is transferred to cryotubes and storedon ice. HEK293 cells are harvested by washing twice with 10 ml coldDulbecco's PBS and lysing by addition of 1.5 ml of CAT lysis buffer(Boehringer Mannheim, Indianapolis, Ind.), followed by incubation for 30min at room temperature. Lysate is transferred to 1.7 ml polypropylenemicrofuge tubes and centrifuged at 1000×g for 10 min. The supernatant istransferred to new cryotubes and stored on ice. Aliquots of cellsupernatants and the lysate of the cells expressing the PS190 proteinconstruct are analyzed for the presence of PS190 recombinant protein.The aliquots can be analyzed using SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), using standard methods and reagents known inthe art. See, e.g., J. Sambrook et al., supra. The gels can then beblotted onto a solid medium such as nitrocellulose, nytran, or the like,and the PS190 protein band can be visualized using western blottingtechniques with anti-myc epitope or anti-histidine monoclonal antibodies(Invitrogen, Carlsbad, Calif.) or PS190 polyclonal serum (see Example14). Alternatively, the expressed PS190 recombinant protein can beanalyzed by mass spectrometry (see Example 12).

D. Purification

Purification of the PS190 recombinant protein containing the myc-hissequence is performed using the Xpress® affinity chromatography system(Invitrogen, Carlsbad, Calif.) containing a nickel-charged agarose resinwhich specifically binds polyhistidine residues. Supernatants from10×100 mm plates, prepared as described supra, are pooled and passedover the nickel-charged column. Non-binding protein is eluted by washingthe column with 50 mM Tris-HCl (pH 7.5)/150 mM NaCl buffer, leaving onlythe myc-his fusion proteins. Bound PS190 recombinant protein then iseluted from the column using either an excess of imidazole or histidine,or a low pH buffer. Alternatively, the recombinant protein can also bepurified by binding at the myc-his sequence to an affinity columnconsisting of either anti-myc or anti-histidine monoclonal antibodiesconjugated through a hydrazide or other linkage to an agarose resin andeluting with an excess of myc peptide or histidine, respectively.

The purified recombinant protein can then be covalently cross-linked toa solid phase, such as N-hydroxysuccinimide-activated sepharose columns(Pharmacia Biotech, Piscataway, N.J.), as directed by supplier'sinstructions. These columns containing covalently linked PS190recombinant protein, can then be used to purify anti-PS190 antibodiesfrom rabbit or mouse sera (see Examples 13 and 14).

E. Coating Microtiter Plates with PS190 Expressed Proteins

Supernatant from a 100 mm plate, as described supra, is diluted in anappropriate volume of PBS. 100 μl of the resulting mixture is placedinto each well of a Reacti-Bind™ metal chelate microtiter plate (Pierce,Rockford, Ill.), incubated at room temperature while shaking, andfollowed by three washes with 200 μl each of PBS with 0.05% Tween® 20.The prepared microtiter plate can then be used to screen polyclonalantisera for the presence of anti-PS190 antibodies (see Example 17).

Although pcDNA3.1/Myc-His is utilized in this example, it is known tothose skilled in the art that other comparable expression systems can beutilized herein with appropriate modifications in reagent and/ortechniques and are within the skill of one of ordinary skill in the art.The largest cloned insert containing the coding region of the PS190 geneis sub-cloned into either (i) a eukaryotic expression vector which maycontain, for example, a cytomegalovirus (CMV) promoter and/or proteinfusible sequences which aid in protein expression and detection, or (ii)a bacterial expression vector containing a superoxide-dismutase (SOD)and CMP-KDO synthetase (CKS) or other protein fusion gene for expressionof the protein sequence. Methods and vectors which are useful for theproduction of polypeptides which contain fusion sequences of SOD aredescribed in European patent application No. EP 0 196 056, publishedOct. 1, 1986, which is incorporated herein by reference, and vectorscontaining fusion sequences of CKS are described in European patentapplication No. EP 0 331 961, published Sep. 13, 1989, which publicationis also incorporated herein by reference. The purified protein can beused in a variety of techniques, including but not limited to, animalimmunization studies, solid phase immunoassays, etc.

Example 12 Chemical Analysis of Prostate Tissue Proteins

A. Analysis of Tryptic Peptide Fragments Using MS

Sera from patients with prostate disease such as prostate cancer, serafrom patients with no prostate disease, extracts of prostate tissues orcells from patients with prostate disease such as prostate cancer,extracts of prostate tissues or cells from patients with no prostatedisease, and extracts of tissues or cells from other non-diseased ordiseased organs of patients, are run on a polyacrylamide gel usingstandard procedures and stained with Coomassie Blue. Sections of the gelsuspected of containing the unknown polypeptide are excised andsubjected to an in-gel reduction, acetamidation and tryptic digestion.P. Jeno et al., Anal. Bio. 224:451-455 (1995) and J. Rosenfeld et al.,Anal. Bio. 203:173-179 (1992). The gel sections are washed with 100 mMNH₄HCO₃ and acetonitrile. The shrunken gel pieces are swollen indigestion buffer (50 mM NH₄HCO₃, 5 mM CaCl₂ and 12.5 μg/ml trypsin) at4° C. for 45 min. The supernatant is aspirated and replaced with 5 to 10μl of digestion buffer without trypsin and allowed to incubate overnightat 37° C. Peptides are extracted with 3 changes of 5% formic acid andacetonitrile and evaporated to dryness. The peptides are adsorbed toapproximately 0.1 μl of POROS R2 sorbent (Perseptive Biosystems,Framingham, Mass.) trapped in the tip of a drawn gas chromatographycapillary tube by dissolving them in 10 μl of 5% formic acid and passingit through the capillary. The adsorbed peptides are washed with waterand eluted with 5% formic acid in 60% methanol. The eluant is passeddirectly into the spraying capillary of an API III mass spectrometer(Perkin-Elmer Sciex, Thornhill, Ontario, Canada) for analysis bynano-electrospray mass spectrometry. M. Wilm et al., Int. J. MassSpectrom. Ion Process 136:167-180 (1994) and M. Wilm et al., Anal. Chem.66:1-8 (1994). The masses of the tryptic peptides are determined fromthe mass spectrum obtained off the first quadrupole. Massescorresponding to predicted peptides can be further analyzed in MS/MSmode to give the amino acid sequence of the peptide.

B. Peptide Fragment Analysis Using LC/MS

The presence of polypeptides predicted from mRNA sequences found inhyperplastic disease tissues also can be confirmed using liquidchromatography/tandem mass spectrometry (LC/MS/MS). D. Hess et al.,METHODS, A Companion to Methods in Enzymology 6:227-238 (1994). Theserum specimen or tumor extract from the patient is denatured with SDSand reduced with dithiothreitol (1.5 mg/ml) for 30 min at 90° C.followed by alkylation with iodoacetamide (4 mg/ml) for 15 min at 25° C.Following acrylamide electrophoresis, the polypeptides areelectroblotted to a cationic membrane and stained with Coomassie Blue.Following staining, the membranes are washed and sections thought tocontain the unknown polypeptides are cut out and dissected into smallpieces. The membranes are placed in 500 μl microcentrifuge tubes andimmersed in 10 to 20 μl of proteolytic digestion buffer (100 mMTris-HCl, pH 8.2, containing 0.1 M NaCl, 10% acetonitrile, 2 mM CaCl₂and 5 μg/ml trypsin) (Sigma, St. Louis, Mo.). After 15 h at 37° C., 3 μlof saturated urea and 1 μl of 100 μg/ml trypsin are added and incubatedfor an additional 5 h at 37° C. The digestion mixture is acidified with3 μl of 10% trifluoroacetic acid and centrifuged to separate supernatantfrom membrane. The supernatant is injected directly onto a microbore,reverse phase HPLC column and eluted with a linear gradient ofacetonitrile in 0.05% trifluoroacetic acid. The eluate is fed directlyinto an electrospray mass spectrometer, after passing though a streamsplitter if necessary to adjust the volume of material. The data isanalyzed following the procedures set forth in Example 12, Section A.

Example 13 Gene Immunization Protocol

A. In Vivo Antigen Expression

Gene immunization circumvents protein purification steps by directlyexpressing an antigen in vivo after inoculation of the appropriateexpression vector. Also, production of antigen by this method may allowcorrect protein folding and glycosylation since the protein is producedin mammalian tissue. The method utilizes insertion of the gene sequenceinto a plasmid which contains a CMV promoter, expansion and purificationof the plasmid and injection of the plasmid DNA into the muscle tissueof an animal. Preferred animals include mice and rabbits. See, forexample, H. Davis et al., Human Molecular Genetics 2:1847-1851 (1993).After one or two booster immunizations, the animal can then be bled,ascites fluid collected, or the animal's spleen can be harvested forproduction of hybridomas.

B. Plasmid Preparation and Purification

PS190 cDNA sequences are generated from the PS190 cDNA-containing vectorusing appropriate PCR primers containing suitable 5′ restriction sitesfollowing the procedures described in Example 11. The PCR product is cutwith appropriate restriction enzymes and inserted into a vector whichcontains the CMV promoter (for example, pRc/CMV or pcDNA3 vectors fromInvitrogen, San Diego, Calif.). This plasmid then is expanded in theappropriate bacterial strain and purified from the cell lysate using aCsCl gradient or a Qiagen plasmid DNA purification column. All thesetechniques are familiar to one of ordinary skill in the art of molecularbiology.

C. Immunization Protocol

Anesthetized animals are immunized intramuscularly with 0.1-100 μg ofthe purified plasmid diluted in PBS or other DNA uptake enhancers(Cardiotoxin, 25% sucrose). See, for example, H. Davis et al., HumanGene Therapy 4:733-740 (1993); and P. W. Wolff et al., Biotechniques11:474-485 (1991). One to two booster injections are given at monthlyintervals.

D. Testing and Use of Antiserum

Animals are bled and the resultant sera tested for antibody usingpeptides synthesized from the known gene sequence (see Example 16) usingtechniques known in the art, such as western blotting or EIA techniques.Antisera produced by this method can then be used to detect the presenceof the antigen in a patient's tissue or cell extract, or in a patient'sserum, by ELISA or Western blotting techniques, such as those describedin Examples 15 through 18.

Example 14 Production of Antibodies Against PS190

A. Production of Polyclonal Antisera

Antiserum against PS190 is prepared by injecting appropriate animalswith peptides whose sequences are derived from that of the predictedamino acid sequence of the PS190 consensus sequence (SEQUENCE ID NO 4).The synthesis of peptides (SEQUENCE ID NO 10, SEQUENCE ID NO 11,SEQUENCE ID NO 12, SEQUENCE ID NO 13, and SEQ ID NO: 14 and fragmentsthereof derived from SEQ ID NO: 9) is described in Example 10. Peptidesused as immunogen either can be conjugated to a carrier such as keyholelimpet hemocyanine (KLH), prepared as described hereinbelow, orunconjugated (i.e., not conjugated to a carrier such as KLH).

1. Peptide Conjugation. Peptide is conjugated to maleimide activatedkeyhole limpet hemocyanine (KLH, commercially available as Inject®,available from Pierce Chemical Company, Rockford, Ill.). Inject containsabout 250 moles of reactive maleimide groups per mole of hemocyanine.The activated KLH is dissolved in phosphate buffered saline (PBS, pH8.4) at a concentration of about 7.7 mg/ml. The peptide is conjugatedthrough cysteines occurring in the peptide sequence, or to a cysteinepreviously added to the synthesized peptide in order to provide a pointof attachment. The peptide is dissolved in dimethyl sulfoxide (DMSO,Sigma Chemical Company, St. Louis, Mo.) and reacted with the activatedKLH at a mole ratio of about 1.5 moles of peptide per mole of reactivemaleimide attached to the KLH. A procedure for the conjugation ofpeptide (SEQUENCE ID NO 10) is provided hereinbelow. It is known to theordinary artisan that the amounts, times and conditions of such aprocedure can be varied to optimize peptide conjugation.

The conjugation reaction described hereinbelow is based on obtaining 3mg of KLH peptide conjugate (“conjugated peptide”), which contains about0.77 μmoles of reactive maleimide groups. This quantity of peptideconjugate usually is adequate for one primary injection and four boosterinjections for production of polyclonal antisera in a rabbit. Briefly,peptide (SEQUENCE ID NO 10) is dissolved in DMSO at a concentration of1.16 μmoles/100 μl of DMSO. One hundred microliters (100 μl) of the DMSOsolution is added to 380 μl of the activated KLH solution prepared asdescribed hereinabove, and 20 μl of PBS (pH 8.4) is added to bring thevolume to 500 μl. The reaction is incubated overnight at roomtemperature with stirring. The extent of reaction is determined bymeasuring the amount of unreacted thiol in the reaction mixture. Thedifference between the starting concentration of thiol and the finalconcentration is assumed to be the concentration of peptide which hascoupled to the activated KLH. The amount of remaining thiol is measuredusing Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid), PierceChemical Company, Rockford, Ill.). Cysteine standards are made at aconcentration of 0, 0.1, 0.5, 2, 5 and 20 mM by dissolving 35 mg ofcysteine HCl (Pierce Chemical Company, Rockford, Ill.) in 10 ml of PBS(pH 7.2) and diluting the stock solution to the desiredconcentration(s). The photometric determination of the concentration ofthiol is accomplished by placing 200 μl of PBS (pH 8.4) in each well ofan Immulon 2® microwell plate (Dynex Technologies, Chantilly, Va.).Next, 10 μl of standard or reaction mixture is added to each well.Finally, 20 μl of Ellman's reagent at a concentration of 1 mg/ml in PBS(pH 8.4) is added to each well. The wells are incubated for 10 minutesat room temperature, and the absorbance of all wells is read at 415 nmwith a microplate reader (such as the BioRad Model 3550, BioRad,Richmond, Calif.). The absorbance of the standards is used to constructa standard curve and the thiol concentration of the reaction mixture isdetermined from the standard curve. A decrease in the concentration offree thiol is indicative of a successful conjugation reaction. Unreactedpeptide is removed by dialysis against PBS (pH 7.2) at room temperaturefor 6 hours. The conjugate is stored at 2-8° C. if it is to be usedimmediately; otherwise, it is stored at −20° C. or colder.

2. Animal Immunization. Female white New Zealand rabbits weighing 2 kgor more are used for raising polyclonal antiserum. Generally, one animalis immunized per unconjugated or conjugated peptide (prepared asdescribed hereinabove). One week prior to the first immunization, 5 to10 ml of blood is obtained from the animal to serve as a non-immuneprebleed sample.

Unconjugated or conjugated peptide is used to prepare the primaryimmunogen by emulsifying 0.5 ml of the peptide at a concentration of 2mg/ml in PBS (pH 7.2) which contains 0.5 ml of complete Freund'sadjuvant (CFA) (Difco, Detroit, Mich.). The immunogen is injected intoseveral sites of the animal via subcutaneous, intraperitoneal, and/orintramuscular routes of administration. Four weeks following the primaryimmunization, a booster immunization is administered. The immunogen usedfor the booster immunization dose is prepared by emulsifying 0.5 ml ofthe same unconjugated or conjugated peptide used for the primaryimmunogen, except that the peptide now is diluted to 1 mg/ml with 0.5 mlof incomplete Freund's adjuvant (IFA) (Difco, Detroit, Mich.). Again,the booster dose is administered into several sites and can utilizesubcutaneous, intraperitoneal and intramuscular types of injections. Theanimal is bled (5 ml) two weeks after the booster immunization and theserum is tested for immunoreactivity to the peptide, as described below.The booster and bleed schedule is repeated at 4 week intervals until anadequate titer is obtained. The titer or concentration of antiserum isdetermined by microtiter EIA as described in Example 17, below. Anantibody titer of 1:500 or greater is considered an adequate titer forfurther use and study.

B. Production of Monoclonal Antibody

1. Immunization Protocol. Mice are immunized using immunogens preparedas described hereinabove, except that the amount of the unconjugated orconjugated peptide for monoclonal antibody production in mice isone-tenth the amount used to produce polyclonal antisera in rabbits.Thus, the primary immunogen consists of 100 μg of unconjugated orconjugated peptide in 0.1 ml of CFA emulsion; while the immunogen usedfor booster immunizations consists of 50 μg of unconjugated orconjugated peptide in 0.1 ml of IFA. Hybridomas for the generation ofmonoclonal antibodies are prepared and screened using standardtechniques. The methods used for monoclonal antibody development followprocedures known in the art such as those detailed in Kohler andMilstein, Nature 256:494 (1975) and reviewed in J. G. R. Hurrel, ed.,Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press,Inc., Boca Raton, Fla. (1982). Another method of monoclonal antibodydevelopment which is based on the Kohler and Milstein method is that ofL. T. Mimms et al., Virology 176:604-619 (1990), which is incorporatedherein by reference.

The immunization regimen (per mouse) consists of a primary immunizationwith additional booster immunizations. The primary immunogen used forthe primary immunization consists of 100 μg of unconjugated orconjugated peptide in 50 μl of PBS (pH 7.2) previously emulsified in 50μl of CFA. Booster immunizations performed at approximately two weeksand four weeks post primary immunization consist of 50 μg ofunconjugated or conjugated peptide in 50 μl of PBS (pH 7.2) emulsifiedwith 50 μl IFA. A total of 100 μl of this immunogen is inoculatedintraperitoneally and subcutaneously into each mouse. Individual miceare screened for immune response by microtiter plate enzyme immunoassay(EIA) as described in Example 17 approximately four weeks after thethird immunization. Mice are inoculated either intravenously,intrasplenically or intraperitoneally with 50 μg of unconjugated orconjugated peptide in PBS (pH 7.2) approximately fifteen weeks after thethird immunization.

Three days after this intravenous boost, splenocytes are fused with, forexample, Sp2/0-Ag14 myeloma cells (Milstein Laboratories, England) usingthe polyethylene glycol (PEG) method. The fusions are cultured inIscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal calfserum (FCS), plus 1% hypoxanthine, aminopterin and thymidine (HAT). Bulkcultures are screened by microtiter plate EIA following the protocol inExample 17. Clones reactive with the peptide used an immunogen andnon-reactive with other peptides (i.e., peptides of PS190 not used asthe immunogen) are selected for final expansion. Clones thus selectedare expanded, aliquoted and frozen in IMDM containing 10% FCS and 10%dimethyl-sulfoxide.

2. Production of Ascites Fluid Containing Monoclonal Antibodies. Frozenhybridoma cells prepared as described hereinabove are thawed and placedinto expansion culture. Viable hybridoma cells are inoculatedintraperitoneally into Pristane treated mice. Ascitic fluid is removedfrom the mice, pooled, filtered through a 0.2μ filter and subjected toan immunoglobulin class G (IgG) analysis to determine the volume of theProtein A column required for the purification.

3. Purification of Monoclonal Antibodies From Ascites Fluid. Briefly,filtered and thawed ascites fluid is mixed with an equal volume ofProtein A sepharose binding buffer (1.5 M glycine, 3.0 M NaCl, pH 8.9)and refiltered through a 0.2μ filter. The volume of the Protein A columnis determined by the quantity of IgG present in the ascites fluid. Theeluate then is dialyzed against PBS (pH 7.2) overnight at 2-8° C. Thedialyzed monoclonal antibody is sterile filtered and dispensed inaliquots. The immunoreactivity of the purified monoclonal antibody isconfirmed by determining its ability to specifically bind to the peptideused as the immunogen by use of the EIA microtiter plate assay procedureof Example 17. The specificity of the purified monoclonal antibody isconfirmed by determining its lack of binding to irrelevant peptides suchas peptides of PS190 not used as the immunogen. The purified anti-PS190monoclonal thus prepared and characterized is placed at either 2-8° C.for short term storage or at −80° C. for long term storage.

4. Further Characterization of Monoclonal Antibody. The isotype andsubtype of the monoclonal antibody produced as described hereinabove canbe determined using commercially available kits (available fromAmersham. Inc., Arlington Heights, Ill.). Stability testing also can beperformed on the monoclonal antibody by placing an aliquot of themonoclonal antibody in continuous storage at 2-8° C. and assayingoptical density (OD) readings throughout the course of a given period oftime.

C. Use of Recombinant Proteins as Immunogens

It is within the scope of the present invention that recombinantproteins made as described herein can be utilized as immunogens in theproduction of polyclonal and monoclonal antibodies, with correspondingchanges in reagents and techniques known to those skilled in the art.

Example 15 Purification of Serum Antibodies Which Specifically Bind toPS190 Peptides

Immune sera, obtained as described hereinabove in Examples 13 and/or 14,is affinity purified using immobilized synthetic peptides prepared asdescribed in Example 10, or recombinant proteins prepared as describedin Example 11. An IgG fraction of the antiserum is obtained by passingthe diluted, crude antiserum over a Protein A column (Affi-Gel proteinA, Bio-Rad, Hercules, Calif.). Elution with a buffer (Binding Buffer,supplied by the manufacturer) removes substantially all proteins thatare not immunoglobulins. Elution with 0.1M buffered glycine (pH 3) givesan immunoglobulin preparation that is substantially free of albumin andother serum proteins.

Immunoaffinity chromatography is performed to obtain a preparation witha higher fraction of specific antigen-binding antibody. The peptide usedto raise the antiserum is immobilized on a chromatography resin, and thespecific antibodies directed against its epitopes are adsorbed to theresin. After washing away non-binding components, the specificantibodies are eluted with 0.1 M glycine buffer, pH 2.3. Antibodyfractions are immediately neutralized with 1.0M Tris buffer (pH 8.0) topreserve immunoreactivity. The chromatography resin chosen depends onthe reactive groups present in the peptide. If the peptide has an aminogroup, a resin such as Affi-Gel 10 or Affi-Gel 15 is used (Bio-Rad,Hercules, Calif.). If coupling through a carboxy group on the peptide isdesired, Affi-Gel 102 can be used (Bio-Rad, Hercules, Calif.). If thepeptide has a free sulfhydryl group, an organomercurial resin such asAffi-Gel 501 can be used (Bio-Rad, Hercules, Calif.).

Alternatively, spleens can be harvested and used in the production ofhybridomas to produce monoclonal antibodies following routine methodsknown in the art as described hereinabove.

Example 16 Western Blotting of Tissue Samples

Protein extracts are prepared by homogenizing tissue samples in 0.1MTris-HCl (pH 7.5), 15% (w/v) glycerol, 0.2 mM EDTA, 1.0 mM1,4-dithiothreitol, 10 μg/ml leupeptin and 1.0 mMphenylmethylsulfonylfluoride (Kain et al., Biotechniques, 17:982(1994)). Following homogenization, the homogenates are centrifuged at 4°C. for 5 minutes to separate supernate from debris. For proteinquantitation, a 3-10 μl volume of supernate is added to 1.5 ml ofbicinchoninic acid reagent (Sigma, St. Louis, Mo.), and the resultingabsorbance at 562 nm is measured.

For SDS-PAGE, samples are adjusted to desired protein concentration withTricine Buffer (Novex, San Diego, Calif.), mixed with an equal volume of2×Tricine sample buffer (Novex, San Diego, Calif.), and heated for 5minutes at 100° C. in a thermal cycler. Samples are then applied to aNovex 10-20% Precast Tricine Gel for electrophoresis. Followingelectrophoresis, samples are transferred from the gels to nitrocellulosemembranes in Novex Tris-Glycine Transfer buffer. Membranes are thenprobed with specific anti-peptide antibodies using the reagents andprocedures provided in the Western Lights or Western Lights Plus(Tropix, Bedford, Mass.) chemiluminesence detection kits.Chemiluminescent bands are visualized by exposing the developedmembranes to Hyperfilm ECL (Amersham, Arlington Heights, Ill.).

Competition experiments are carried out in an analogous manner as above,with the following exception; the primary antibodies (anti-peptidepolyclonal antisera) are pre-incubated for 30 minutes at roomtemperature with varying concentrations of peptide immunogen prior toexposure to the nitrocellulose filter. Development of the Western isperformed as above.

After visualization of the bands on film, the bands can also bevisualized directly on the membranes by the addition and development ofa chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate(BCIP). This chromogenic solution contains 0.016% BCIP in a solutioncontaining 100 mM NaCl, 5 mM MgCl₂ and 100 mM Tris-HCl (pH 9.5). Thefilter is incubated in the solution at room temperature until the bandsdevelop to the desired intensity. Molecular mass determination is madebased upon the mobility of pre-stained molecular weight standards(Novex, San Diego, Calif.) or biotinylated molecular weight standards(Tropix, Bedford, Mass.).

Example 17 EIA Microtiter Plate Assay

The immunoreactivity of antiserum preferably obtained from rabbits ormice as described in Example 13 or Example 14 is determined by means ofa microtiter plate EIA, as follows. Synthetic peptides prepared asdescribed in Example 10, or recombinant proteins prepared as describedin Example 11, are dissolved in 50 mM carbonate buffer (pH 9.6) to afinal concentration of 2 μg/ml. Next, 100 μl of the peptide or proteinsolution is placed in each well of an Immulon 2® microtiter plate (DynexTechnologies, Chantilly, Va.). The plate is incubated overnight at roomtemperature and then washed four times with deionized water. The wellsare blocked by adding 125 μl of a suitable protein blocking agent, suchas Superblock®(Pierce Chemical Company, Rockford, Ill.), in phosphatebuffered saline (PBS, pH 7.4) to each well and then immediatelydiscarding the solution. This blocking procedure is performed threetimes. Antiserum obtained from immunized rabbits or mice prepared aspreviously described is diluted in a protein blocking agent (e.g., a 3%Superbloc® solution) in PBS containing 0.05% Tween-20® (monolauratepolyoxyethylene ether) (Sigma Chemical Company, St. Louis, Mo.) and0.05% sodium azide at dilutions of 1:500, 1:2500, 1:12,500, 1:62,500 and1:312,500 and placed in each well of the coated microtiter plate. Thewells then are incubated for three hours at room temperature. Each wellis washed four times with deionized water. A 100 μl volume of alkalinephosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse IgGantiserum (Southern Biotech, Birmingham, Ala.), diluted 1:2000 in 3%Superblock® solution in phosphate buffered saline containing 0.05% Tween20® and 0.05% sodium azide, is added to each well. The wells areincubated for two hours at room temperature. Next, each well is washedfour times with deionized water. One hundred microliters (100 μl) ofparanitrophenyl phosphate substrate (Kirkegaard and Perry Laboratories,Gaithersburg, Md.) then is added to each well. The wells are incubatedfor thirty minutes at room temperature. The absorbance at 405 nm is readof each well. Positive reactions are identified by an increase inabsorbance at 405 nm in the test well above that absorbance given by anon-immune serum (negative control). A positive reaction is indicativeof the presence of detectable anti-PS190 antibodies.

In addition to titers, apparent affinities [K_(d)(app)] may also bedetermined for some of the anti-peptide antisera. EIA microtiter plateassay results can be used to derive the apparent dissociation constants(K_(d)) based on an analog of the Michaelis-Menten equation (V. VanHeyningen, Methods in Enzymology, Vol.121, p. 472 (1986) and furtherdescribed in X. Qiu et al., Journal of Immunology, Vol. 156, p. 3350(1996)):$\left\lbrack {{Ag} - {Ab}} \right\rbrack = {\left\lbrack {{Ag} - {Ab}} \right\rbrack_{\max} \times \frac{\lbrack{Ab}\rbrack}{\lbrack{Ab}\rbrack = K_{d}}}$

Wherein [Ag-Ab] is the antigen-antibody complex concentration,[Ag-Ab]_(max) is the maximum complex concentration, [Ab] is the antibodyconcentration, and K_(d) is the dissociation constant. During the curvefitting, the [Ag-Ab] is replaced with the background subtracted value ofthe OD_(405nm) at the given concentration of Ab. Both K_(d) and[OD_(405nm)]_(max), which corresponds to the [Ag-Ab]_(max), are treatedas fitted parameters. The software program Origin™ can be used for thecurve fitting.

Example 18 Coating of Solid Phase Particles

A. Coating of Microparticles with Antibodies Which Specifically Bind toPS190 Antigen

Affinity purified antibodies which specifically bind to PS190 protein(see Example 15) are coated onto microparticles of polystyrene,carboxylated polystyrene, polymethylacrylate or similar particles havinga radius in the range of about 0.1 to 20 pm. Microparticles may beeither passively or actively coated. One coating method comprisescoating EDAC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (Aldrich Chemical Co., Milwaukee, Wis.) activatedcarboxylated latex microparticles with antibodies which specificallybind to PS190 protein, as follows. Briefly, a final 0.375% solidsuspension of resin washed carboxylated latex microparticles (availablefrom Bangs Laboratories, Carmel, IN or Serodyn, Indianapolis, Ind.) aremixed in a solution containing 50 mM MES buffer, pH 4.0 and 150 mg/l ofaffinity purified anti-PS190 antibody (see Example 14) for 15 min in anappropriate container. EDAC coupling agent is added to a finalconcentration of 5.5 μg/ml to the mixture and mixed for 2.5 h at roomtemperature.

The microparticles then are washed with 8 volumes of a Tween 20®/sodiumphosphate wash buffer (pH 7.2) by tangential flow filtration using a 0.2μm Microgon Filtration module. Washed microparticles are stored in anappropriate buffer which usually contains a dilute surfactant andirrelevant protein as a blocking agent, until needed.

B. Coating of ¼ Inch Beads

Antibodies which specifically bind to PS190-antigen also may be coatedon the surface of ¼ inch polystyrene beads by routine methods known inthe art (Snitman et al., U.S. Pat. No. 5,273,882, incorporated herein byreference) and used in competitive binding or EIA sandwich assays.

Polystyrene beads first are cleaned by ultrasonicating them for about 15seconds in 10 mM NaHCO₃ buffer at pH 8.0. The beads then are washed indeionized water until all fines are removed. Beads then are immersed inan antibody solution in 10 mM carbonate buffer, pH 8 to 9.5. Theantibody solution can be as dilute as 1 μg/ml in the case of highaffinity monoclonal antibodies or as concentrated as about 500 μg/ml forpolyclonal antibodies which have not been affinity purified. Beads arecoated for at least 12 hours at room temperature, and then they arewashed with deionized water. Beads may be air dried or stored wet (inPBS, pH 7.4). They also may be overcoated with protein stabilizers (suchas sucrose) or protein blocking agents used as non-specific bindingblockers (such as irrelevant proteins, Carnation skim milk, Superblock®,or the like).

Example 19 Microparticle Enzyme Immunoassay (MEIA)

PS190 antigens are detected in patient test samples by performing astandard antigen competition EIA or antibody sandwich EIA and utilizinga solid phase such as microparticles (MEIA). The assay can be performedon an automated analyzer such as the IMx® Analyzer (Abbott Laboratories,Abbott Park, Ill.).

A. Antibody Sandwich EIA

Briefly, samples suspected of containing PS190 antigen are incubated inthe presence of anti-PS190 antibody-coated microparticles (prepared asdescribed in Example 17) in order to form antigen/antibody complexes.The microparticles then are washed and an indicator reagent comprisingan antibody conjugated to a signal generating compound (i.e., enzymessuch as alkaline phosphatase or horseradish peroxide) is added to theantigen/antibody complexes or the microparticles and incubated. Themicroparticles are washed and the bound antibody/antigen/antibodycomplexes are detected by adding a substrate (e.g., 4-methylumbelliferyl phosphate (MUP), or OPD/peroxide, respectively), thatreacts with the signal generating compound to generate a measurablesignal. An elevated signal in the test sample, compared to the signalgenerated by a negative control, detects the presence of PS190 antigen.The presence of PS190 antigen in the test sample is indicative of adiagnosis of a prostate disease or condition, such as prostate cancer.

B. Competitive Binding Assay

The competitive binding assay uses a peptide or protein that generates ameasurable signal when the labeled peptide is contacted with ananti-peptide antibody coated microparticle. This assay can be performedon the IMx® Analyzer (available from Abbott Laboratories, Abbott Park,Ill.). The labeled peptide is added to the PS190 antibody-coatedmicroparticles (prepared as described in Example 17) in the presence ofa test sample suspected of containing PS190 antigen, and incubated for atime and under conditions sufficient to form labeled PS190 peptide (orlabeled protein)/bound antibody complexes and/or patient PS190antigen/bound antibody complexes. The PS190 antigen in the test samplecompetes with the labeled PS190 peptide (or PS190 protein) for bindingsites on the microparticle. PS190 antigen in the test sample results ina lowered binding of labeled peptide and antibody coated microparticlesin the assay since antigen in the test sample and the PS190 peptide orPS190 protein compete for antibody binding sites. A lowered signal(compared to a control) indicates the presence of PS190 antigen in thetest sample. The presence of PS190 antigen suggests the diagnosis of aprostate disease or condition, such as prostate cancer.

The PS190 polynucleotides and the proteins encoded thereby which areprovided and discussed hereinabove are useful as markers of prostatetissue disease, especially prostate cancer. Tests based upon theappearance of this marker in a test sample such as blood, plasma orserum can provide low cost, non-invasive, diagnostic information to aidthe physician to make a diagnosis of cancer, to help select a therapyprotocol, or to monitor the success of a chosen therapy. This marker mayappear in readily accessible body fluids such as blood, urine or stoolas antigens derived from the diseased tissue which are detectable byimmunological methods. This marker may be elevated in a disease state,altered in a disease state, or be a normal protein of the prostate whichappears in an inappropriate body compartment.

21 1 187 DNA Homo sapiens n = a or g or c or t/u, unknown or other atposition 2 1 tnttttttgt ttgctctgaa tttattgcga gtgaaaaaca gagaaaatcctcaagtttaa 60 gtttctgata gcagagtgtg ggagttagag catggngagt ccagaggttccagaccccca 120 aaggtctcta ccagggccat ctccgttagt ggcggtggca gcccctcttgtggccttttt 180 cctctct 187 2 250 DNA Homo sapiens 2 gtttgctctgaatttattgc gagtgaaaaa cagagaaaat cctcaagttt aagtttctga 60 tagcagagtgtgggagttag agcatgggga gtccagaggt tccagacccc caaaggtctc 120 taccagggccatctccgtta gtggcggtgg cagcccctct tgtggccttt ttcctctctc 180 caaggggtcaccccgcacca tgccgctccc cctcatctat cttgccccct ccccacaggc 240 ccatctgcgc250 3 224 DNA Homo sapiens n = a or g or c or t/u, unknown or other atposition 71 3 cgatcgggca agtaaacccc ctccctcgcc gacttcggaa ctggcgagagttcagcgcag 60 atgggcctgt ngngangggg naagatagat ganggggagc ggcatggtgcggggtgaccc 120 cttggagaga ggaaaaangc cacaagaggg gctgccaccg ccactaacggagatggccct 180 ggtaganact nngggggtct ggaactnctg gnctccccat gcnc 224 4312 DNA Homo sapiens n = a or g or c or t/u, unknown or other atposition 2 4 tnttttttgt ttgctctgaa tttattgcga gtgaaaaaca gagaaaatcctcaagtttaa 60 gtttctgata gcagagtgtg ggagttagag catggggagt ccagaggttccagaccccca 120 aaggtctcta ccagggccat ctccgttagt ggcggtggca gcccctcttgtggccttttt 180 cctctctcca aggggtcacc ccgcaccatg ccgctccccc tcatctatcttgccccctcc 240 ccacaggccc atctgcgctg aactctcgcc agttccgaag tcggcgagggagggggttta 300 cttgcccgat cg 312 5 68 DNA Homo sapiens 5 agctcggaattccgagcttg gatcctctag agcggccgcc gactagtgag ctcgtcgacc 60 cgggaatt 68 668 DNA Homo sapiens 6 aattaattcc cgggtcgacg agctcactag tcggcggccgctctagagga tccaagctcg 60 gaattccg 68 7 24 DNA Homo sapiens 7 agcggataacaatttcacac agga 24 8 18 DNA Homo sapiens 8 tgtaaaacga cggccagt 18 9 73PRT Homo sapiens 9 Met Gly Ser Pro Glu Val Pro Asp Pro Gln Arg Ser LeuPro Gly Pro 1 5 10 15 Ser Pro Leu Val Ala Val Ala Ala Pro Leu Val AlaPhe Phe Leu Ser 20 25 30 Pro Arg Gly His Pro Ala Pro Cys Arg Ser Pro SerSer Ile Leu Pro 35 40 45 Pro Pro His Arg Pro Ile Cys Ala Glu Leu Ser ProVal Pro Lys Ser 50 55 60 Ala Arg Glu Gly Val Tyr Leu Pro Asp 65 70 10 24PRT Homo sapiens 10 Met Gly Ser Pro Glu Val Pro Asp Pro Gln Arg Ser LeuPro Gly Pro 1 5 10 15 Ser Pro Leu Val Ala Val Ala Ala 20 11 31 PRT Homosapiens 11 Leu Val Ala Phe Phe Leu Ser Pro Arg Gly His Pro Ala Pro CysArg 1 5 10 15 Ser Pro Ser Ser Ile Leu Pro Pro Pro His Arg Pro Ile CysAla 20 25 30 12 21 PRT Homo sapiens 12 Ser Pro Arg Gly His Pro Ala ProCys Arg Ser Pro Ser Ser Ile Leu 1 5 10 15 Pro Pro Pro His Arg 20 13 29PRT Homo sapiens 13 Ser Ile Leu Pro Pro Pro His Arg Pro Ile Cys Ala GluLeu Ser Pro 1 5 10 15 Val Pro Lys Ser Ala Arg Glu Gly Val Tyr Leu ProAsp 20 25 14 13 PRT Homo sapiens 14 Val Pro Lys Ser Ala Arg Glu Gly ValTyr Leu Pro Asp 1 5 10 15 8 PRT Homo sapiens 15 Asp Tyr Lys Asp Asp AspAsp Lys 1 5 16 21 PRT Homo sapiens 16 Glu Gln Lys Leu Ile Ser Glu GluAsp Leu Asn Met His Thr Glu His 1 5 10 15 His His His His His 20 17 246DNA Homo sapiens 17 tttttgtttg ctctgaattt attgcgagtg aaaaacagagaaaatcctca agtttaagtt 60 tctgatagca gagtgtggga gtagagcatg gggagtccagaggttccaga cccccaaagg 120 tctctaccag gggcatctcc gttagtggcg gtggcagcccctcttgtggc ctttttcctc 180 tctccaaggg gtcaccccgc accatgccgc tccccctcatctatcttgcc ccctccccac 240 aggcca 246 18 704 DNA Homo sapiens 18tttttgtttg ctctgaattt attgcgagtg aaaaacagag aaaatcctca agtttaagtt 60tctgatagca gagtgtggga gttagagcat ggggagtcca gaggttccag acccccaaag 120gtctctacca gggccatctc cgttagtggc ggtggcagcc cctcttgtgg cctttttcct 180ctctccaagg ggtcaccccg caccatgccg ctccccctca tctatcttgc cccctcccca 240caggcccatc tgcgctgaac tctcgccagt tccgaagtcg gcgagggagg gggtttactt 300gcccgatcgt tggtgggttt gagcttatag aggcagagga gtaagaacct gcgatattga 360aagctaccca catggggctt ccttgaagga ggacgtggaa ggcagaaagt gacctgctct 420gagcggcgca tgtaaccgag gaccttaagc tggaccacgg ggcttggacg attttttaaa 480tcaggaaatc gacctcatct tcctcctcct cgtcctcttc ccctgaaccc ccagtccgca 540tgcactcaca ctctttggcc ttttccctca gtcccgggct cctctttggt aaatagattt 600gtaggtgtct aagtcacgtc ccaccctcac tccttcccag gagaggagac agggctagga 660tcccacccga ccgcgggcca taaacacttg gctgcggcgg ccgc 704 19 263 DNA Homosapiens 19 gaggaagatg aggtcgattt cctgatttaa aaaatcgtcc aagccccgtggtccagctta 60 aggtcctcgg ttacatgcgc cgctcagagc aggtcacttt ctgccttccacgtcctcctt 120 caaggaagcc ccatgtgggt agctttcaat atcgcaggtt cttactcctctgcctctata 180 agctcaaacc caccaacgat cgggcaagta aaccccctcc ctcgccgacttcggaactgg 240 cgagagttca gcgcagatgg gcc 263 20 203 DNA Homo sapiens n =a or g or c or t/u, unknown or other at position 16 20 gaggagcccgggactnaggg aaaaggccaa agagtgtgag tgcatgcgga ctgggggttc 60 aggggaagaggacgaggngg aggaagatga ggtcgatttc ctgatttaaa aaatcgtcca 120 agccccgtggtccagcttaa ggtcctcggt tacatgcgcc gctcagagca ggtcactttc 180 tgccttccacgtcctccttc aag 203 21 157 DNA Homo sapiens 21 gcccgcggtc gggtgggatcctagccctgt ctcctctcct gggaaggagt gagggtggga 60 cgtgacttag acacctacaaatctatttac caaagaggag cccgggactg agggaaaagg 120 ccaaagagtg tgagtgcatgcggactgggg gttcagg 157

We claim:
 1. A method for detecting an antigen in a test samplesuspected of containing said antigen, comprising: (a) contacting thetest sample with an antibody which specifically binds to at least oneepitope of a polypeptide selected from the group consisting of: fullcomplements of SEQ ID NOS: 9-14, so that antibody/antigen complexes areformed; and (b) detecting the presence of said complexes as anindication of the presence of said antigen.
 2. The method of claim 1,wherein said antibody is attached to a solid phase.
 3. A method fordetecting an antigen in a test sample suspected of containing saidantigen, comprising: (a) contacting the test sample with an antibodywhich specifically binds to at least one epitope of a polypeptideselected from the group consisting of SEQ ID NOS: 9-13, so thatantibody/antigen complexes are formed; and (b) detecting the presence ofsaid complexes as an indication of the presence of said antigen.
 4. Themethod of claim 3, wherein said antibody is attached to a solid phase.5. A method for detecting an antigen in a test sample suspected ofcontaining said antigen, comprising: (a) contacting the test sample withan antibody which specifically binds to at least one epitope of apolypeptide selected from the group consisting of: SEQ ID NOS: 10-13 andfragments thereof, so that antibody/antigen complexes are formed; and(b) detecting the presence of said complexes as an indication in thepresence of said PS190 antigen.
 6. The method of claim 5, wherein saidantibody is attached to a solid phase.